Compositions of vaccines and adjuvants and methods for the treatment of urinary tract infections

ABSTRACT

This invention describes novel adjuvant compositions and formulations with excellent stability at refrigerated and room temperatures and up to and about 37° C. that can be produced at remarkably low costs. This invention describes novel vaccine compositions and formulations to treat and prevent urinary tract infections caused by gram-negative bacteria including  Escherichia coli  and multi-drug resistant  E. coli . This invention also describes methods of administration of said novel vaccine compositions and formulations and methods of treatment to prevent and treat urinary tract infections caused by gram-negative bacteria including  E. coli  and multi-drug resistant  E. coli.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.14/494,001, which claims benefit of U.S. Provisional Application No.61/882,498 filed on Sep. 25, 2013, the contents of which areincorporated in its entirety by reference.

The Sequence Listing in the ASCII text file named Sequence_ST25.txtcreated on Apr. 3, 2015, which is 4.45 KB, is incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention provides novel adjuvant compositions and formulations withexcellent stability at refrigerated and room temperatures, and also upto about 37° C., that can be produced at remarkably low costs. Thesenovel adjuvant compositions and formulations are used in vaccines andexhibit superior properties of enhancing immune responses to antigenswhile causing less severe injection site and systemic reactions. Theinvention also describes novel vaccine compositions and formulations totreat and prevent urinary tract infections caused by gram-negativebacteria including Escherichia coli and multi-drug resistant E. coli.The invention also provides methods of administration of said novelvaccine formulations and methods of treatment to prevent and treaturinary tract infections caused by gram-negative bacteria including E.coli and multi-drug resistant E. coli.

2. Description of the Related Art

In the United States (US) and other countries most populations areprotected from numerous infectious diseases by the use of vaccines.Vaccines protect people from infectious diseases such as diphtheria,tetanus, pertussis, hepatitis, influenza, and polio to name a few.Society relies on the protections afforded by vaccines, which haverendered most of these infectious diseases only a part of history forthe citizens of the US. In fact, in the US, the Centers for DiseaseControl (CDC) administer the Vaccines for Children program whichprovided free vaccinations for approximately 40 million children in2010. Approximately 70% of these children are enrolled in Medicaid. Toprevent outbreaks of disease and reduce the costs of treating theseinfectious diseases, the US has made vaccination a national priorityindependent of economic status. Low-cost vaccines are desperatelyrequired and these vaccines are a national priority now and in theforeseeable future.

Given the importance of vaccines, the need to continually develop newand improved vaccines to improve the health of our population is clear.Even more critical is the need to provide lower cost vaccines to assistwith reducing the skyrocketing costs of the US healthcare system. Anational priority is to lower the costs of the US healthcare system.

-   -   Contributing to these difficulties, even in the US, is        compliance with vaccine storage requirements. A study conducted        by the Office of the Inspector General at the Department of        Health and Human Services (HHS) and reported in 2012        (OEI-04-10-00430) found that providers participating in the        Vaccines for Children Program of the CDC: 1.) exposed vaccines        to temperatures outside their approved temperature ranges; 2.)        stored vaccines in refrigerators and freezers at temperatures        outside their approved temperature ranges; and 3.) had expired        vaccines stored with non-expired vaccines.

Another issue with vaccines is that vaccines can have a short shelf lifeand are prone to expire prior to use.

In addition, the study described above conducted by the Office of theInspector General found that 16 of 46 US healthcare providers ofVaccines for Children program had expired vaccines stored with unexpiredvaccines. On average, these expired vaccines had been expired for about6 months. For example, it was reported that as of Jul. 1, 2010 40million unused doses of swine flu vaccine that cost about $260 millionto produce had just expired and were being destroyed. Vaccineexpirations result in significant economic losses each year in the US.

Adjuvants enhance the immune responses to antigens of vaccines. Of the34 vaccines provided under the Vaccines for Children Programadministered by the CDC in the US, 20 contain adjuvants. Of these 20vaccines with adjuvants, 19 of these vaccines contain alum adjuvants and1 vaccine contains monophosphoryl lipid A adsorbed to alum (GSK's MPL)as the adjuvant.

Despite industry wide attempts at developing new adjuvants, currentlyonly alum and GSK's MPL are used in approved vaccines in the US.Numerous adjuvant development failures have occurred in the US, but theneed for new and effective adjuvants remains high.

GlaxoSmithKline's (GSK) Cervarix vaccine containing3′-O-desacyl-4′-monophosphoryl lipid A adsorbed to alum (GSK's MPL) waslicensed in the US for the prevention of cervical cancer caused by humanpapillomavirus. Because the starting material to produce MPL is isolatedfrom Salmonella minnesota, the final product is a dynamic, complexmixture of hexa-, penta-, and tetraacyl analogues; each of theseanalogues differ in biological activity. As a result, the mixture of3′-O-desacyl-4′-monophosphoryl lipid A presents manufacturing, testing,and use challenges that greatly contribute to the expense and supplyissues with the vaccine.

In addition to storage problems, vaccine injections are often painful tothe recipient. Redness, swelling, itching and tenderness at injectionsites may occur after administration of a vaccine. The PrescribingInformation of GSK's Cervarix Vaccine containing MPL and alum adjuvantslists local adverse events that may include pain, redness, and swelling.Local pain that prevented activities of daily life was reported inapproximately 8 percent of subjects receiving either GSK's Cervarixvaccine or the adjuvant alum alone. Systemic adverse reactions observedafter administration of vaccines containing MPL and alum adjuvantsinclude headache, fatigue, fever, rash, myalgia, arthralgia, urticaria,and gastrointestinal symptoms including nausea, vomiting, diarrhea,and/or abdominal pain.

Furthermore, as described in the “Clinical Review of HumanPapillomavirus Bivalent (Types 16 and 18) vaccine [GSK's CervarixVaccine], Recombinant, Biologics License Application EfficacySupplement” four studies reported local adverse events including localpain preventing movement in approximately 16 percent of subjects.Swelling was also reported at greater than 50 mm in approximately 3percent of subjects. The same four studies reported systemic severeadverse events in 2.4 to 7.8 percent for arthralgia, fatigue,gastrointestinal, headache, and myalgia.

The severity of the injection site reactions and systemic reactions aresignificant requiring medical treatment involving narcotic use, IVhydration, or other physician implemented treatments and loss of workfrom preventing daily activity due to diarrhea, myalgia, fatigue,headache, and vomiting.

The Advisory Committee on Immunization Practices establishesrecommendations for the National Strategy for Pandemic Influenza. Thisstrategy includes the need to “provide pandemic vaccine to all UScitizens within 6 months of a pandemic declaration: pandemic vaccine(600 million doses) [National Strategy for Pandemic Influenza (November2005) and HHS Pandemic Influenza Plan (November 2005)” and requires theuse of adjuvants to attempt to move toward this seemingly unapproachablevaccination target. Since there is no approved adjuvant for a generalflu vaccine in the US, the US national vaccine stockpile was without analternative and purchased the MF59 adjuvant from Novartis for about $500million. The MF59 adjuvant was recently discontinued in a clinical studyof Fluad Paediatric due to “high vaccine reactogenicity observed inchildren 9 through 12 years of age, the protocol of study V7P29 wasamended to exclude children less than 9 years of age.” The evidencesupports that during a national pandemic declaration a significantnumber of severe reactions will occur due to the use of the MF59adjuvant and require additional medical care.

A synthetic analogue of monophosphoryl lipid A was introduced by AvantiPolar Lipids (Alabaster, Ala., USA) in around 2004 time period. AvantiPolar Lipids named this synthetic analogue phosphorylated hexaacyldisaccharide PHAD, alternatively known as “GLA”. phosphorylated hexaacyldisaccharide supplied by Avanti Polar Lipids is provided as a singlecompound, shown in FIG. 1, of approximately 98% purity with a molecularweight of 1763 Daltons. PHAD's purity is in stark contrast to GSK's MPLisolated from Salmonella minnesota that, as described above, exists as adynamic, complex mixture. Unlike GSK's MPL, PHAD's manufacturingprocess, supply, use, and stability can be closely monitored andcontrolled as a pure compound.

Whether or not a specific adjuvant or combination of adjuvants willenhance an immune response toward each specific antigen isunpredictable. For example, GSK's Cervarix vaccine contains bothmonophosphoryl lipid A and alum, because this combination is superior toalum alone (Giannini et al. Vaccine, 2006, 24, p. 5937-5949). A similarincrease in efficacy was observed with a vaccine that used a recombinanthepatitis B surface antigen. (Vaccine, 1998, 16(7), p. 708-714). Anotherexample demonstrating the variability of antigen—adjuvant combinationsin producing an immune response for a specific antigen is shown in Table6 of U.S. Pat. No. 6,889,885. These inventors demonstrated that theQS-21 adjuvant and, separately, the alum plus monophosphoryl lipid Aadjuvant combination generated greater antibody responses to a 74 kDprotein than alum or monophosphoryl lipid A alone. Furthermore, in 2009Derek T. O'Hagan and Ennio De Gregorio of Novartis Vaccines published areview about the development of adjuvants. (Drug Discovery Today,14(11/12), June 2009, p. 541-551) They reported that alum is arelatively weak adjuvant for certain proteins or antigens and newadjuvants are still required.

In 2004 the Infectious Disease Society of America (IDSA) forewarned apending crisis of increasing antibiotic resistant bacteria throughoutthe world with no new antibiotics on the horizon to combat thisoccurrence. In 2009 the IDSA identified that bacterial infections nowoccur that are resistant to all current antibiotics, and that the mostalarming antibiotic resistant bacteria are gram-negative bacteriaincluding E. coli. In 2010, the IDSA stated that despite efforts by manyprivate, public, and government laboratories, research had not producedany new alternatives to treat antibiotic resistant bacteria and a globalcommitment was now required. IDSA's urgency is supported by scientistsat GlaxoSmithKline who predicted it would be greater than ten to fifteenyears prior to the launch of any new antibiotics for the treatment ofgram-negative bacterial infections (Payne et al. Nature Reviews DrugDiscovery. 2007, 6, p. 29-40).

Their prediction was based upon the failure of 34 companies thatattempted to develop new antibiotics. A consensus among the scientificcommunity is emerging that the US urgently needs new treatments forbacterial infections. Adam L. Hersh and colleagues reported a surveywith 562 infectious disease physicians responding across the US in thejournal of Clinical Infectious Disease in 2012 (Hersh et al. CID. 2012.54(11), 1677-8) that 63% of these physicians had treated patients withbacterial infections resistant to all known antibiotics within the lastyear. These data emphasize the need for new treatments for bacterialinfections. The failure in the art to identify new therapeuticalternatives to prevent and treat gram-negative bacterial infections iswell documented.

Moreover, at least five vaccines under development to prevent or treatStaphylococcus aureus infections have recently been discontinued. Theseinclude STAPHVAX, Veronate, Aurexis, Aurograb, and V710. The failure toidentify new vaccines to prevent and treat bacterial infections is welldocumented.

Urinary tract infections (UTIs) are one of the most prevalent infectiousdiseases worldwide and the number one infectious disease suffered bywomen in the US. Symptoms of UTIs include dysuria (painful urination),urgency to urinate, and suprapubic pain. Acute uncomplicated UTIs occurin an estimated 7 to 11 million women in the US each year. Over half ofall adult women will suffer from one or more UTIs in their lifetime with25-44% of women experiencing a recurrent UTI. In fact, approximately1,000,000 women and men in the US experience three or more UTI episodesper year. Moreover, recurrence often occurs within 30 to 90 days ofinfection despite appropriate antibiotic treatment and apparentclearance of the initial infection from the urine.

Despite recent progress in the epidemiology and pathogenesis of UTI,there have been no recent major improvements in our ability to actuallyprevent or treat these infections. The 25 to 44% of women with UTI whoexperience recurrent infections require additional treatment, additionalcosts, and in some cases extensive urological evaluation to prevent moresevere complications from arising. Thus, safe and effective vaccinesthat have the potential to improve patient convenience and decreasecosts are of considerable interest to patients, providers, and healthcare organizations. In the recurrent UTI population, antimicrobialresistance is of great concern since treatment options are diminishing.There is, therefore, an urgent need to develop new approaches to UTIprevention and treatment that depend less on the use of antimicrobials.

UTIs are most commonly caused by uropathogenic Escherichia coli (UPEC),which can be responsible for up to 85% of community-acquired UTIs. Acritical pathogenic cascade by which UPEC evade host defenses andrapidly expand in numbers in the urinary tract to cause disease has beenuncovered. This work supports the clinical need for a UTI vaccine.

FimH plays a significant role in several stages of the pathogenesiscascade, which makes it a prime vaccine target. UPEC strains that lackthe FimH adhesin are unable to effectively colonize the bladder. Avaccine against FimH will activate host defenses to recognize and clearUPEC at all stages of infection, even when protected in IBCs orintracellular reservoirs.

A FimCH vaccine with MF59 as the adjuvant containing squalene wasjointly invented by scientists at MedImmune, Inc. and the laboratory ofProfessor Scott Hultgren (U.S. Pat. No. 6,500,434; incorporated hereinin its entirety). The FimH protein and FimC protein exist as anon-covalent protein complex, FimCH. FimC stabilizes FimH and antibodiesare produced against both proteins, however, only antibodies to FimHhave been shown to reduce E. coli colonization of bladders in animals.The use of FimCH as an antigen in a vaccine is therefore limited by therequirement of an effective adjuvant.

The FimCH vaccine with the MF59 adjuvant containing squalene (anoil-in-water emulsion) elicited an immune response during Phase 1clinical trials (United States Patent Application 20030138449;incorporated herein in its entirety.). Phase 2 clinical trials wereconducted in two distinct populations again with the MF59 adjuvantcontaining squalene, but women did not produce relevant IgG titers toFimH in either of these trials. The development of MedImmune's FimCHvaccine with the MF59 adjuvant was discontinued because of thesedisappointing results. The MF59 adjuvant with squalene has a history ofcausing severe local injection site and systemic reactions when usedwith certain antigens. During these Phase 2 clinical trials, womenexperienced severe injection site reactions and severe systemicreactions. Because of this failure, a vaccine for the treatment orprevention of UTI does not exist in the US.

An ongoing need exists for a vaccine to prevent and treat UTI. Thefailure of MedImmune and others to develop a UTI vaccine evidences thedifficulties of developing new vaccines for bacterial infections.MedImmune demonstrated that alum does not sufficiently enhance theimmune response to FimCH. MedImmune had no clear alternatives ofadjuvants to pair with the FimCH antigen.

Accordingly, there is an urgent need for vaccines, and adjuvants used toenhance the immune response to antigens in vaccines. There is a need forvaccines and adjuvants for vaccines that have extended stability withoutsacrificing efficacy. In particular, there is an urgent and widelyrecognized need in for more room temperature stable vaccines andadjuvants. In addition, it would be desirable to have vaccines,adjuvants and compositions that are stable at temperature above roomtemperature.

In addition there is a need for adjuvants and pharmaceuticalcompositions that produce less severe injection site and systemicreactions.

There is a need for new vaccines to prevent and treat bacterialinfections, and for vaccines for the prevention and treatment of UT's inparticular.

It would be desirable to have vaccines, and adjuvants used to enhancethe immune response of antigens in vaccines, with increased shelf-livesthat can be produced in a cost effective manner. Such vaccines andadjuvants would significantly lower healthcare costs in the US,particularly if they can be stored at room temperature or greaterwithout negatively affecting their stability.

It would be desirable to have adjuvants and vaccines that produceminimal injection site and systemic reactions. It would be desirable tohave formulations with as few as excipients as possible.

It would be desirable to have a vaccine, and adjuvant for a vaccine thatenhances the immune response treat bacterial infections. It would bedesirable to have a vaccine, and adjuvant for a vaccine that enhancesthe immune response to Escherichia coli to patients with UTI.

BRIEF SUMMARY OF THE INVENTION

The present invention addresses many problems of prior art adjuvants,vaccines and pharmaceutical compositions described herein. The presentinvention provides novel liquid adjuvant compositions and formulationswhich provides many unexpected and advantageous properties unknown inthe art of adjuvant and pharmaceutical compositions.

In one aspect, liquid adjuvant compositions and formulations thatexhibit room temperature stability for about more than 6 months and upto about 37° C. for about 60 or more days is provided. The novel liquidadjuvant compositions and formulations can be stored at refrigerated orroom temperature conditions facilitating its shelf life during shippingand storage and lowering its delivery costs.

The adjuvant formulations of the invention described herein address manycurrent obstacles in vaccine administration by enabling a low cost andunexpectedly and remarkably stable adjuvant formulation which enhancesan immune response to E. coli antigen with less severe injection siteand systemic reactions.

The data described herein demonstrate that the adjuvant formulations ofthe invention enhance the immune response to other antigens includingbacterial and viral antigens.

The invention described herein contributes to reducing this problem bytreating urinary tract infections caused by gram-negative bacteriaincluding E. coli.

In one aspect, a novel adjuvant composition with remarkable stability at2° C. to 8° C. and room temperature up to about 37° C. is disclosed.

In one aspect of the invention, a composition comprising onesynthetically produced adjuvant phosphorylated hexaacyl disaccharide anda buffer selected from the group consisting of citrate, succinate, andphosphate at about 25 mM to about 50 mM, preferably 28 mM to about 50mM, and most preferably 30 mM to about 50 mM. These novel phosphorylatedhexaacyl disaccharide compositions are preferably aqueous bufferedsuspensions. The composition can be used in a variety of ways in thevaccine and pharmaceutical context. The composition, with preferably noadditional components, significantly improves the stability ofphosphorylated hexaacyl disaccharide in suspension and achievesexceptional stability at room temperature and up to and at about 37° C.The compositions also exhibit excellent stability at refrigeratedtemperature as well. This represents a significant advancement inadjuvant and pharmaceutical technology by providing an efficient andeconomical phosphorylated hexaacyl disaccharide composition that doesnot require refrigeration for long term stability.

In another aspect of the invention, novel adjuvant formulations as anaqueous buffered suspension are provided. In one embodiment the adjuvantformulations include one synthetically produced adjuvant phosphorylatedhexaacyl disaccharide, a buffer selected from the group consisting ofcitrate, succinate, and phosphate at about 10 mM to about 50 mM,preferably about 25 mM to about 50 mM, more preferably 28 mM to about 50mM, and most preferably 30 mM to about 50 mM, and preferably onesynthetically produced phosphatidylcholine. When a phosphatidylcholineis added, the preferred buffer concentrations can be expanded to about10 mM to about 50 mM and achieve the remarkable stability describedherein. These novel adjuvant formulations are preferably aqueousbuffered suspensions. The adjuvant formulations have excellent long-termstability when stored at refrigerated and room temperatures andexcellent stability up to and at about 37° C. These formulations can beproduced at remarkably low costs.

The novel adjuvant formulations described herein do not requirelyophilization, or equivalent process for room temperature stability orstability up to and at about 37° C. The adjuvant formulations include aspecific buffer and optionally and preferably one or more syntheticallyproduced phosphatidylcholines selected from the group consisting ofDMPC, DPPC, DSPC, DOPC, and POPC, preferably DPPC, and one syntheticallyproduced adjuvant, phosphorylated hexaacyl disaccharide, in a molarratio of about 1:1 to 40:1 (phosphatidylcholine:phosphorylated hexaacyldisaccharide), preferably about 1:1 to 20:1(phosphatidylcholine:phosphorylated hexaacyl disaccharide), morepreferably about 2:1 to 5:1 (phosphatidylcholine:phosphorylated hexaacyldisaccharide), and most preferably about 2:1 to 5:1 (DPPC:phosphorylatedhexaacyl disaccharide).

One of the most valuable aspects of the invention is that the adjuvantformulations include only a single adjuvant phosphorylated hexaacyldisaccharide in citrate, succinate or phosphate buffers at specifiedconcentrations as described herein, and preferably a singlephosphatidylcholine. No other ingredients are required to produce theunexpected long-term stability at room temperature. Further the adjuvantformulation can be produced at low cost. In this regard, the long-termstability of these adjuvant formulations at room temperature isremarkable and is achieved without the use of cholesterol,phosphatidylglycerol, phosphatidylethanolamine, monoacylglycerol,lyoprotectants, and metabolizable oil. Conventional prior art adjuvantsdo not achieve stability without the use of one or more of theseingredients.

Another aspect of this invention is adjuvant formulations that do notneed two or more phosphatidylcholines or the addition of aphosphatidylglycerol. As shown in the examples, two or morephosphatidylcholines or one or more phosphatidylglycerols can be addedto these formulations, but preferably it is not needed to achieve theremarkable long-term stability demonstrated herein.

While not bound by theory, expansion of the preferred bufferconcentrations of citrate, succinate, or phosphate buffer to about 10 mMto about 50 mM to achieve the remarkable stability of the inventiondescribed herein is believed to be due to addition of preferredexcipients, preferably phosphatidylcholine, at the defined molar ratioof the preferred aspect of the invention of phosphatidylcholine tophosphorylated hexaacyl disaccharide described herein. More preferably,the preferred buffer concentrations are about 25 mM to about 50 mM, evenmore preferably 28 mM to about 50 mM, and most preferably 30 mM to about50 mM. Preferably the pH is in a range of about 4.0 to about 7.5,preferably about 4.5 to about 6.5, more preferably about 5.0 to 6.0.

As shown in the examples, this exceptional stability at room temperatureis not present when formulated in water, acetate buffer, PBS, or citrateor phosphate buffers at or greater than 100 mM. Instead the stability isproduced by citrate, succinate, or phosphate at concentrations of about10 mM to about 50 mM, but preferably about 25 mM to about 50 mM, morepreferably 28 mM to about 50 mM, and most preferably 30 mM to about 50mM.

Another embodiment of the invention includes a non-ionic surfactant,preferably polysorbate 80, to reduce the aggregation of particles of theinvention.

Removing the need for lyophilization is a significant advantage andunexpected breakthrough because many costly steps and risks have beeneliminated. Another aspect of this invention is that these adjuvantformulations are superior at enhancing an immune response to an antigenwhile causing significantly less severe injection site and systemicreactions during administration compared to the prior art. Anotheraspect of this invention is adjuvant formulations substantially free ofmetabolizable oils, including squalene, and substantially free ofcholesterol. In the art, it is widely considered that cholesterol isnecessary for adjuvant formulations or liposomes to function. Thecurrent invention produces all its benefits as described herein withoutrequiring cholesterol.

Therefore, the advantages of the adjuvant formulations andpharmaceutical compositions described herein include: room temperaturestability as an aqueous buffered suspension for at least 6 months,and/or stability up to and at about 37° C. for about 60 or more days;less severe injection site and systemic reactions per administrationwhile enhancing immune responses to antigens; and, lower cost ofproduction or manufacturing with fewer materials or components and lowerconcentration of the materials or components. The inventive adjuvantformulations provide these three combined major benefits not previouslyachieved by adjuvants that are synthetic analogues of MLA or MPL asalternatives to alum-based adjuvants.

In another aspect of the invention novel vaccine compositions containingthe novel adjuvant formulations for use to treat and prevent urinarytract infections caused by gram-negative bacteria including Escherichiacoli and multi-drug resistant E. coli are provided. Methods ofadministration of said novel vaccine compositions and methods oftreatment to prevent and treat urinary tract infections caused bygram-negative bacteria including E. coli and multi-drug resistant E.coli are also provided.

In another aspect of the invention methods of inducing the production ofantibodies against FimH in a human with recurrent urinary tractinfections are provided.

Another aspect of the invention is vaccine compositions that induce theproduction of antibodies against FimH in a human with recurrent urinarytract infections.

In another aspect of the invention, a vaccine kit comprising thephosphorylated hexaacyl disaccharide compositions or formulations orvaccine compositions, along with administration directions andinstructions for storage are provided. The instructions provide for theexposure of the phosphorylated hexaacyl disaccharide compositions orformulations at room temperature and up to and about 37° C. Theseinstructions describing storage, shipping, and exposure temperatures maybe approved by a government regulatory authority including the US FDA orEuropean Medicines Agency. Preferably, one or more of the kit componentsis the phosphorylated hexaacyl disaccharide compositions or formulationsin a syringe.

Another aspect of the invention is that the phosphorylated hexaacyldisaccharide composition and formulations and vaccine compositions aresterile compositions and sterile pharmaceutical compositions, morepreferably the sterile phosphorylated hexaacyl disaccharide compositionsand formulations are contained in a sterile syringe.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical structure of one salt of phosphorylatedhexaacyl disaccharide

FIG. 2 shows the chemical structure of DPPC

FIG. 3 is a graph illustrating the indirect ELISA of FimCH and Q133Kusing IgG anti-FimH.

FIG. 4 is a graph illustrating the potency assay analyzing FimCH andQ133K.

FIG. 5 is a graph illustrating the evaluation of small moleculeinhibitors in the potency assay. Two small molecules,4-methylumbelliferyl-α-D-mannopyranoside (UFMP) andmethyl-α-D-mannopyranoside (MDMP) inhibit mannose binding to FimH.

FIG. 6 is a representative chromatogram of the FimCH drug substancesample by CEX-HPLC.

FIG. 7 is a graph illustrating the protection from E. coli Infectionfollowing FimCH/phosphorylated hexaacyl disaccharide immunization ofmice.

FIG. 8 is an example chromatogram of DPPC and phosphorylated hexaacyldisaccharide by HPLC.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“About” in reference to phosphorylated hexaacyl disaccharide quantity orbuffer concentration (unless defined) means plus and minus 10% of thelisted quantity.

“About 25° C.” refers to temperatures at 20° C. to 30° C.

“About 37° C.” refers to temperatures at 34° C. to 40° C.

“About 50 mM” refers to the buffer concentrations described herein meansbetween 50 mM and less than 100 mM. As shown in the examples, 100 mM orgreater of the specified buffers is not as effective in enablinglong-term room temperature stability. The upper end of bufferconcentrations is typically evaluated at two-fold increments. Withreference to about 50 mM, the specified buffer concentrations of theinvention are preferably less than 90 mM, preferably less than 80 mM,more preferably less than 70 mM, and most preferably less than 60 mM.

“About 10 mM” refers to the buffer concentrations described herein meansbetween 6 mM and 10 mM.

“Acceptable carrier” refers to a carrier that is not deleterious to theother ingredients of the composition and is not deleterious to materialto which it is to be applied.

“Adjuvant” refers to an agent that, when present in an effective amount,increases the antigenic response; a substance enhancing the immuneresponse to an antigen; or an agent that stimulates antibody productionto an antigen. Numerous naming conventions or terminologies exist in theart. Without reference to a specific naming convention, the adjuvantcompositions as described herein may simply be referred to as adjuvantformulations or adjuvant preparations.

“Administration” refers to any means of providing a compound orcomposition to a subject.

“Colloid” refers to one or more chemicals, compounds, or substancesmicroscopically dispersed throughout an aqueous buffered solution oranother substance. The adjuvant formulations described herein can alsobe described as colloid. One example of a colloidal dispersion isFungizone, which consists of Amphotericin B-sodium desoxycholate forparenteral administration.

“Critical micelle concentration” refers to the concentration ofsurfactant(s) above which micelles form and all additional surfactantsadded to the system go to micelles.

“DLPC” refers to 1,2-dilauroyl-sn-glycero-3-phosphocholine.

“DMPC” refers to 1,2-dimyristoyl-sn-glycero-3-phosphocholine.

“DOPC” refers to 1,2-dioleoyl-sn-glycero-3-phosphocholine.

“DPPC” refers to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (molecularformula C₄₀H₈₀NO₈P (MW=734 Da) (the chemical structure is shown in FIG.2).

“DPPG” refers to 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol).

“DSPC” refers to 1,2-distearoyl-sn-glycero-3-phosphocholine.

“Effective amount” refers to a sufficient amount of FimCH or truncatedFimH or other antigen in a vaccine composition that is administered to ahuman to elicit an immune response against FimH or other antigen, orsufficient amount of an adjuvant, preferably phosphorylated hexaacyldisaccharide, to elicit an increased immune response to antigen.

“Essentially free” in reference to materials, additives, chemicals, orexcipients means the materials, additives, chemicals, or excipients havenot been added to the composition or formulation of the invention,although some impurity level amounts may be present.

“Essentially free of severe injection site and systemic reactions” meanstwo percent or less of humans experience these severe injection site andsystemic reactions that are attributable to the adjuvant composition orformulation.

“Injection site reaction” refers to pain, tenderness, redness, and/orswelling at the site of administration or injection site.

“Invention” means at least some embodiments of the present invention;references to various feature(s) of the “invention” or “presentinvention” throughout this document do not mean that all claimedembodiments or methods include the referenced feature(s).

“Labeling” or “Label” refers to all labels and other written, printed,or graphic matter upon any article or any of its containers or wrappers,or accompanying such article and, therefore, includes any packageinserts or information sheets that accompany vaccine or adjuvantcompositions or formulations of the invention.

“Less severe injection site and systemic reactions” refers to lesssevere or Grade 3 injection site reactions and/or systemic reactions ascompared to commercial vaccine Cervarix as detailed herein and in itsproduct information documents and investigational vaccine adjuvantGLA-SE as described herein and in Treanor et al. (Vaccine 2013).

“Liposome” refers to generally vesicles that consist of a lipid bilayermembrane surrounding a hydrophilic core.

“Low cost” or “low costs” refers to a composition with the components ormaterials at the lowest concentrations sufficient to achieve the novelcharacteristics of the invention.

“Lyoprotectants” refers to materials, chemicals, or excipients primarilyused to protect materials from freezing damage or other impairmentduring manufacturing, storage and use or improving reconstitutionincluding enabling appropriate solvation prior to use, and also includesthese materials, chemicals, or excipients used to modify osmolality oradjust tonicity, and include but not limited to sorbitol, mannitol,mannose, erythritol, xylitol, glycerol, sucrose, dextrose, trehalose,maltose, lactose, and cellobiose.

“Metabolizable oil” refers primarily to squalene, or closely relatedanalogues of squalene as used as an adjuvant in vaccine formulations oradjuvant formulations, but also refers to medium-chain triglyceridesincluding Miglyol 810 and oils from vegetables, animals, or fish whenused in vaccine or adjuvant formulations as excipients, or to create anadjuvant effect, or to produce emulsions. Examples include grapeseedoil, soybean oil, coconut oil, olive oil, sunflower oil, corn oil, andshark liver oil.

“Micelle” refers to an aggregate of surfactant molecules dispersed in anaqueous buffered solution with the hydrophilic head regions in contactwith surrounding aqueous buffered solution, sequestering the hydrophobicsingle-tail regions in the center of the micelle.

“MLA” refers to monophosphoryl lipid A.

“MPL” refers to 3′-O-desacyl-4′-monophosphoryl lipid A.

“Pharmaceutically acceptable carrier” refers to a carrier that is notdeleterious to the other ingredients of the composition and is notdeleterious to the human or other animal recipient thereof. In thecontext of the other ingredients of the composition, “not deleterious”means that the carrier will not react with or degrade the otheringredients or otherwise interfere with their efficacy. Interferencewith the efficacy of an ingredient does not, however, refer to meredilution of the ingredient.

“Phosphorylated hexaacyl disaccharide” is a Toll-like receptor 4 agonistand refers to phosphorylated hexaacyl disaccharide or pharmaceuticallyacceptable salts of phosphorylated hexaacyl disaccharide. The structureof a preferred phosphorylated hexaacyl disaccharide salt is shown inFIG. 1, which is available from Avanti Polar Lipids (PHAD).Phosphorylated hexaacyl disaccharide, as used herein may be fully orpartially synthetic or non-synthetic, although fully synthetic ispreferred.

“Pharmaceutical composition” refers to a composition given to a mammalintended to treat or prevent a disease, or in the case of a vaccinecomposition, to produce an immunogenic response that treats or preventsa disease, reduce symptoms, or provides some type of therapeuticbenefit, or in the case of an adjuvant composition, to enhance an immuneresponse to one or more antigens.

“Phosphate buffer” or “phosphate” refers to a phosphate buffer selectedfrom the following group: sodium phosphate dibasic, sodium phosphatemonobasic, potassium phosphate monobasic, and potassium phosphatedibasic, or some combination thereof. Preferably “phosphate” consists ofsodium phosphate dibasic, sodium phosphate monobasic, and potassiumphosphate monobasic. Unless otherwise noted, reference to phosphatebuffer specifically excludes ammonium phosphate.

“PBS” refers to phosphate-buffered saline of a general composition ofphosphate buffer (Na₂HPO₄ and/or KH₂PO₄), potassium chloride and sodiumchloride. A typical PBS composition is comprised of about 10 mMphosphate buffer (Na₂HPO₄ and/or KH₂PO₄), 2.7 mM potassium chloride and0.14 M sodium chloride, pH 7.4, at 25° C.

“Phosphate citrate buffer” refers to a phosphate buffer containingcitric acid and sodium phosphate where the pH is maintained bycitrate/citric acid and phosphate/hydrogen phosphate equilibrium. Thephosphate may include, for example Na₂HPO₄ and/or KH₂PO₄ and trisodiumcitrate may be used.

“Phosphatidylcholine” (alternatively referred to as “PC”) refers tolipids containing choline. Examples include, but not limited to, DMPC(1,2-dimyristoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), DOPC(1,2-dioleoyl-sn-glycero-3-phosphocholine), and POPC(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Phosphatidylcholinesare available from Avanti Polar Lipids.

“Phosphatidylethanolamine” refers to lipids containing a phosphate groupattached to an ethanolamine, e.g.1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

“Phosphatidylglycerol” refers to lipids containing glycerol. Examplesinclude, but are not limited to,1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG),1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG), and1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG).

“POPC” refers to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

“Recurrent urinary tract infections” refers to a human has 3 to 4Urinary Tract Infections in approximately one year.

“Refrigerated” refers to a temperature range from 2° C. to 8° C.

“Room temperature” refers to a range of temperature from 19° C. (66° F.)to 25° C. (77° F.).

“Saline” refers to about 125 mM to about 155 mM of NaCl in bufferedaqueous solutions. For example, PBS generally contains 137 mM NaCl andTris buffered saline may contain 150 mM.

“Severe injection site reaction” refers to one or more of the following:pain requiring narcotic pain reliever or that prevents daily activity;tenderness causing significant discomfort at rest; redness of more than10 cm; and swelling of more than 10 cm or prevents daily activity.

“Severe systemic reaction” refers to one or more of the following:nausea/vomiting which prevents daily activity or requires outsubject IVhydration; diarrhea consisting of 6 or more watery stools or >800 gramswith 24 hours or requires outsubject IV hydration; headache consistingof significant use of narcotic pain reliever or prevents daily activity;fatigue consisting of significant or prevents daily activity; andmyalgia consisting of significant or prevents daily activity.

“Substantially free” in referring to cholesterol means that cholesterol,if present, is at 0.3 mM or less.

“Substantially free” in referring to monoacylglycerol means,monoacylglycerol, if present, is at 0.5 mM or less. An example ofmonoacylglycerol is monopalmitoyl glycerol.

“Substantially free” in referring to phosphatidylglycerol orphosphatidylethanolamine means these substances, if present, are at 0.1mM or less.

“Substantially free” in referring to lyoprotectants means thesesubstances, if present, are at a concentration of the composition orformulation of 0.5% or less.

“Substantially free of saline” means less than 30 mM NaCl in thecomposition or formulation of the invention.

“Systemic reactions” refers to nausea/vomiting, diarrhea, headache,fatigue, and/or myalgia.

“Succinate buffer” or “succinate” refers to disodium succinate or sodiumsuccinate dibasic. Potassium succinate may be used, but is lesspreferred.

“Stability” or “stable” in reference to adjuvants, actives, proteins,antigens, or drugs refers to the quality of the substance or product toremain acceptable for its intended use throughout a certain time periodbeginning from its date of manufacture while under the influence of suchvariables as temperature and/or humidity. The stability of a substanceis often demonstrated by analytical data (or other equivalent evidence).

“Trisodium citrate” refers to citrate buffers (also referred to as“citrate”) such as, for example, trisodium citrate dihydrate, sodiumcitrate, sodium citrate tribasic hydrate, or citric acid trisodium saltdihydrate as referred to by suppliers including Sigma-Aldrich and BDHChemicals. Potassium citrate, sodium citrate monobasic, and sodiumcitrate dibasic may be used, but they are less preferred. For example,the product imiglucerase for injection uses a combination of trisodiumcitrate and disodium hydrogen citrate. These types of combinations areacceptable. Citric acid, CAS 77-92-9, may be used to adjust the pH ofthe buffer, but it cannot substitute for the listed buffers herein.

“Truncated FimH” refers to the FimH protein truncated to include atleast about 25 to about 175 amino acid residues from the first 175 aminoacids of FimH. With reference to truncated FimH, the FimH proteintruncated to include preferably at least 9% of the FimH protein, morepreferably at least 30% of the FimH protein, and most preferably atleast 60% of the FimH protein.

“Urinary Tract Infections” refers to a medical diagnosis characterizedby 1 or more of the following signs and symptoms: irritative voidingsuch as frequency, urgency, and dysuria; gross hematuria; or elicitedsuprapubic tenderness upon examination; and/or 1 or more of thefollowing laboratory results: positive urine dipstick test from cleancatch or catheter urine specimen; microscopic urinalysis from cleancatch or catheter urine specimen (leukocytes, bacteria, and casts may bepresent); or urine culture from clean catch or catheter urine specimenfor E. coli at 10³ CFU/mL.

“Vaccine” or “vaccine composition” refers to a composition that improvesimmunity to a disease. The vaccine compositions are immunogeniccompositions that elicit immune responses and antibody production towardthe antigen of the composition.

Embodiments of the Invention

In one embodiment a pharmaceutical composition or pharmaceuticallyacceptable carrier is provided. The pharmaceutical compositions orpharmaceutically acceptable carriers are comprised of phosphorylatedhexaacyl disaccharide and a buffer (referred to as “phosphorylatedhexaacyl disaccharide composition” or “phosphorylated hexaacyldisaccharide containing composition” or “phosphorylated hexaacyldisaccharide containing composition and carriers”). The phosphorylatedhexaacyl disaccharide compositions of the invention are preferablyaqueous buffered suspensions. The buffer is selected from the groupconsisting of citrate, succinate, and phosphate at about 25 mM to about50 mM, more preferably 28 mM to about 50 mM, and most preferably 30 mMto about 50 mM. Preferably the pH is in a range of about 4.0 to about7.5, preferably about 4.5 to about 6.5, more preferably about 5.0 toabout 6.0. The pharmaceutical composition or carrier with thiscombination (phosphorylated hexaacyl disaccharide and the buffer)demonstrates excellent stability as a base for pharmaceuticalcompositions and improves the overall stability of phosphorylatedhexaacyl disaccharide compositions. Specifically these phosphorylatedhexaacyl disaccharide containing compositions and carriers achievestability at room temperature and up to about 37° C. Thesephosphorylated hexaacyl disaccharide containing compositions andcarriers of the present invention also exhibit excellent long-termstability at refrigerated temperatures to room temperature.

The phosphorylated hexaacyl disaccharide containing pharmaceuticalcompositions and pharmaceutical carriers (which as previously describedinclude phosphorylated hexaacyl disaccharide and a specific buffer) mayoptionally include other ingredients typical to vaccines, adjuvantformulations, and other pharmaceutical compositions such as excipients,modifiers, surfactants, and additives. For one example,phosphatidylcholines (as described in more detail below) may optionallybe added, alone or in combination with other lipid carriers. In oneembodiment naturally derived phosphatidylcholines from soy or egg orhydrogenated phosphatidylcholines from soy or egg or synthetic ornatural mixed acyl phosphatidylcholines may be added.

In a preferred embodiment, one or more vaccine antigens are added to thephosphorylated hexaacyl disaccharide containing compositions to form avaccine. The vaccine antigens may be any antigen used in vaccinesincluding, but not limited to diphtheria, tetanus, pertussis,poliomyelitis, hepatitis, and or antigenic preparations of the influenzavirus. Preferably the vaccine antigen is a FimCH protein complex asdescribed in detail below.

The phosphorylated hexaacyl disaccharide containing compositions andcarriers can be prepared at any concentration but are typically preparedat about 0.005 to about 1.0 mg/ml of phosphorylated hexaacyldisaccharide, preferably about 0.05 to 1.0 mg/ml of phosphorylatedhexaacyl disaccharide, but usually not more than about 2.5 mg/ml ofphosphorylated hexaacyl disaccharide.

The phosphorylated hexaacyl disaccharide containing compositions may beadministered to animals or humans as an adjuvant of vaccines topreferably deliver about 10 microgram of phosphorylated hexaacyldisaccharide per dose to about 50 micrograms of phosphorylated hexaacyldisaccharide per dose. The exact dose may be modified according to theantigen used. More preferably about 20 micrograms of phosphorylatedhexaacyl disaccharide per dose to about 50 micrograms of phosphorylatedhexaacyl disaccharide per dose is administered. Even more preferablyabout 40 micrograms of phosphorylated hexaacyl disaccharide per dose toabout 50 micrograms of phosphorylated hexaacyl disaccharide per dose isadministered.

As shown herein, the unexpected stability of the phosphorylated hexaacyldisaccharide containing composition at room temperature and up to 37° C.is absent when formulated in water, acetate buffer, PBS, or citrate orphosphate buffers at or greater than 100 mM. The phosphorylated hexaacyldisaccharide compositions contain citrate, succinate, or phosphatebuffer to produce the unexpected stability, preferably about 25 mM toabout 50 mM, more preferably 28 mM to about 50 mM, and most preferably30 mM to about 50 mM.

More particularly, preferred buffers of the phosphorylated hexaacyldisaccharide composition, the concentrations of the buffers are selectedfrom the group consisting of about 25 mM to about 50 mM; 25 mM to about50 mM; about 30 mM to about 50 mM; 28 mM to about 50 mM; 30 mM to about50 mM, about 30 mM; about 40 mM to about 50 mM; 40 mM to about 50 mM;about 40 mM; and about 50 mM.

In addition, as shown in the examples below, the use of PBS in thephosphorylated hexaacyl disaccharide containing compositions andformulations do not exhibit the novel characteristics of the invention,and in particularly the stability characteristics of the invention. Incontrast, the use of citrate, succinate, and phosphate buffers asspecifically defined herein enables the novel stability characteristicsof the invention.

The phosphorylated hexaacyl disaccharide compositions of the inventionare preferably essentially free of squalene.

The phosphorylated hexaacyl disaccharide compositions of the inventionare preferably essentially free of metabolizable oil used as anadjuvant.

The phosphorylated hexaacyl disaccharide compositions of the inventionare preferably essentially free of metabolizable oil.

The phosphorylated hexaacyl disaccharide compositions of the inventionare preferably aqueous buffered suspensions, and preferably theseaqueous buffered suspensions have particle sizes less than 150 nm, evenmore preferably less than 130 nm, and preferably these phosphorylatedhexaacyl disaccharide compositions are not oil-in-water emulsions.

The phosphorylated hexaacyl disaccharide compositions of the inventionare preferably essentially free of a second adjuvant including alum,squalene, QS21, MF59, Toll-like receptor 9 agonists, and other adjuvantsincluding squalene-based adjuvants. Second adjuvants have the potentialto generate more severe local and systemic reactions in humans yetwithout the benefit of further increasing an immune response to improvetherapeutic outcomes.

The phosphorylated hexaacyl disaccharide compositions preferably containless than 5 mM of cholesterol, more preferably less than 1 mM ofcholesterol, and even more preferably substantially free of cholesterol,and most preferably essentially free of cholesterol.

Preferably, the phosphorylated hexaacyl disaccharide compositions aresubstantially free of phosphatidylglycerol, and more preferably areessentially free of phosphatidylglycerol.

Preferably, the phosphorylated hexaacyl disaccharide compositions aresubstantially free of phosphatidylethanolamine, and more preferablyessentially free of phosphatidylethanolamine.

Preferably, the phosphorylated hexaacyl disaccharide compositions aresubstantially free of monoacylglycerol, and more preferably essentiallyfree of monoacylglycerol.

The phosphorylated hexaacyl disaccharide compositions are preferablysubstantially free of saline, preferably contain less than 20 mM NaCl,and more preferably contain less than 10 mM NaCl, and even morepreferably essentially free of NaCl.

The phosphorylated hexaacyl disaccharide compositions are preferablysubstantially free of lyoprotectants, and even more preferablyessentially free of lyoprotectants.

The phosphorylated hexaacyl disaccharide compositions do not requirelyophilization, or equivalent process, to preserve the concentration ofphosphorylated hexaacyl disaccharide for shelf-life or stability.Therefore, the phosphorylated hexaacyl disaccharide compositions arepreferably not lyophilized or lyophilization does not occur; thephosphorylated hexaacyl disaccharide compositions are preferably notdried after preparation; the phosphorylated hexaacyl disaccharidecompositions preferably do not require reconstitution from driedmaterial with liquid or buffer after preparation; and the phosphorylatedhexaacyl disaccharide compositions preferably remain as an aqueousbuffered suspension after manufacturing prior to administration.

Preferably, the phosphorylated hexaacyl disaccharide compositions aresubstantially free of cholesterol, phosphatidylglycerol, andphosphatidylethanolamine, and essentially free of metabolizable oils anda second adjuvant.

More preferably, the phosphorylated hexaacyl disaccharide compositionsare essentially free of cholesterol, phosphatidylglycerol, andphosphatidylethanolamine, and essentially free of metabolizable oils anda second adjuvant.

Preferably the phosphorylated hexaacyl disaccharide compositions of theinvention are substantially free of materials or excipients as describedherein that are not required to achieve the novel characteristics of theinvention, but even more preferably the phosphorylated hexaacyldisaccharide compositions of the invention are essentially free ofmaterials or excipients as described herein that are not required toachieve the novel characteristics of the invention.

Preferably the phosphorylated hexaacyl disaccharide compositions of theinvention are substantially free of one, two, three, or more ofmaterials or excipients per composition as described herein, and thislimitation is not exclusive to only one material or excipient percomposition.

Preferably the phosphorylated hexaacyl disaccharide compositions of theinvention are essentially free of one, two, three, or more materials orexcipients per composition as described herein, and this limitation isnot exclusive to only one material or excipient per composition.

Preferably, when a phosphorylated hexaacyl disaccharide containingcomposition or formulation of the invention is stored, shipped, held, oradministered at room temperature or up to about 37° C., it is mostpreferred to have been sterile filtered or prepared using steriletechniques, more preferably the sterile phosphorylated hexaacyldisaccharide compositions and formulations are contained in a sterilesyringe.

In another embodiment novel adjuvant formulations are provided. Theadjuvant formulations include one synthetically produced phosphorylatedhexaacyl disaccharide, a buffer selected from the group consisting ofcitrate, succinate, and phosphate at about 10 mM to about 50 mM, andpreferably one synthetically produced phosphatidylcholine (referred toas “phosphorylated hexaacyl disaccharide formulations” or“phosphorylated hexaacyl disaccharide containing adjuvant formulations”or “phosphorylated hexaacyl disaccharide containing formulations”). Thephosphatidylcholine and phosphorylated hexaacyl disaccharide are presentat a molar ratio of about 1 (PC) to 1 (phosphorylated hexaacyldisaccharide) to about 40 (PC) to 1 (phosphorylated hexaacyldisaccharide), preferably about 2.5 (PC) to 1 (phosphorylated hexaacyldisaccharide). Reference to phosphorylated hexaacyl disaccharideformulations apply to the adjuvant formulations unless otherwise noted.These novel phosphorylated hexaacyl disaccharide formulations arepreferably aqueous buffered suspensions. The adjuvant formulations haveexcellent long-term stability when stored at refrigerated temperaturesto room temperatures, and up to about 37° C. Additionally, the adjuvantformulations can be produced at low costs.

In a preferred embodiment, the adjuvant formulations include preferablyone synthetically produced phosphatidylcholine, preferably DPPC, and onesynthetically produced adjuvant phosphorylated hexaacyl disaccharide, ina molar ratio of about 1:1 to 40:1 (DPPC:phosphorylated hexaacyldisaccharide), and citrate, succinate, or phosphate buffers at about 10mM to 50 mM, but preferably about 25 mM to 50 mM, more preferably 28 mMto about 50 mM, and most preferably 30 mM to about 50 mM. Preferably thepH is in a range of about 4.0 to about 7.5, preferably about 4.5 toabout 6.5, more preferably about 5.0 to about 6.0. Importantly, thephosphorylated hexaacyl disaccharide adjuvant formulations can beproduced with only one adjuvant phosphorylated hexaacyl disaccharide andpreferably one phosphatidylcholine, thereby enabling them to be producedat low cost. Preferably, the adjuvant formulations containphosphorylated hexaacyl disaccharide as the sole adjuvant, howeveradditional adjuvants may be used. The long-term stability attribute ofthe invention further reduces costs of using these phosphorylatedhexaacyl disaccharide containing adjuvant formulations.

While not bound by theory, expansion of the preferred bufferconcentrations of citrate, succinate, or phosphate buffer to about 10 mMto about 50 mM in the phosphorylated hexaacyl disaccharide formulationsto achieve the remarkable stability of the invention described herein isbelieved to be due to addition of a preferred excipient, preferablyphosphatidylcholine, at the defined molar ratio of the preferred aspectof the invention phosphatidylcholine to phosphorylated hexaacyldisaccharide described herein.

The phosphorylated hexaacyl disaccharide containing adjuvantformulations of the invention are preferably essentially free ofsqualene.

The phosphorylated hexaacyl disaccharide containing adjuvantformulations of the invention are preferably essentially free ofmetabolizable oil used as an adjuvant.

The phosphorylated hexaacyl disaccharide containing adjuvantformulations of the invention are preferably essentially free ofmetabolizable oils.

The phosphorylated hexaacyl disaccharide containing adjuvantformulations of the invention are preferably aqueous bufferedsuspensions, and preferably these aqueous buffered suspensions haveparticle sizes less than 150 nm, even more preferably less than 130 nm,and preferably these phosphorylated hexaacyl disaccharide containingadjuvant formulations are not oil-in-water emulsions.

The phosphorylated hexaacyl disaccharide containing adjuvantformulations of the invention are preferably essentially free of asecond adjuvant including alum, squalene, QS21, MF59, Toll-like receptor9 agonists, and other adjuvants including squalene based adjuvants. Thephosphorylated hexaacyl disaccharide formulations are preferablyessentially free of other or additional adjuvants because they have thepotential to generate more severe local and systemic reactions in humanswithout the benefit of further increasing an immune response orsubstantially improving therapeutic outcomes.

The phosphorylated hexaacyl disaccharide containing adjuvantformulations preferably contain less than 5 mM of cholesterol, morepreferably less than 1 mM of cholesterol, even more preferablysubstantially free of cholesterol, and most preferably essentially freeof cholesterol.

Preferably, the phosphorylated hexaacyl disaccharide containing adjuvantformulations are substantially free of phosphatidylglycerol, and morepreferably essentially free of phosphatidylglycerol.

Preferably, the phosphorylated hexaacyl disaccharide containing adjuvantformulations are substantially free of phosphatidylethanolamine, andmore preferably essentially free of phosphatidylethanolamine.

Preferably, the phosphorylated hexaacyl disaccharide containing adjuvantformulations are substantially free of monoacylglycerol, and morepreferably essentially free of monoacylglycerol.

The phosphorylated hexaacyl disaccharide containing adjuvantformulations of the invention are preferably substantially free ofsaline, preferably contain less than 20 mM NaCl, and more preferably,the phosphorylated hexaacyl disaccharide formulations contain less than10 mM NaCl, and even more preferably are essentially free of NaCl.

Preferably, the phosphorylated hexaacyl disaccharide containing adjuvantformulations are substantially free of lyoprotectants, and morepreferably essentially free of lyoprotectants.

Preferably, the phosphorylated hexaacyl disaccharide containing adjuvantformulations are substantially free of cholesterol,phosphatidylglycerol, and phosphatidylethanolamine, and essentially freeof metabolizable oils and a second adjuvant.

More preferably, the phosphorylated hexaacyl disaccharide containingformulations are essentially free of cholesterol, phosphatidylglyceroland phosphatidylethanolamine, and essentially free of metabolizable oilsand a second adjuvant.

Preferably the phosphorylated hexaacyl disaccharide containingformulations of the invention are substantially free of materials orexcipients as described herein that are not required to achieve thenovel characteristics of the invention, but even more preferably thephosphorylated hexaacyl disaccharide containing formulations of theinvention are essentially free of materials or excipients as describedherein that are not required to achieve the novel characteristics of theinvention.

Preferably the phosphorylated hexaacyl disaccharide containingformulations of the invention are substantially free of one, two, three,or more of materials or excipients per phosphorylated hexaacyldisaccharide containing formulation as described herein, and thislimitation is not exclusive to only one material or excipient perphosphorylated hexaacyl disaccharide containing formulation.

Preferably the phosphorylated hexaacyl disaccharide containingformulations of the invention are essentially free of one, two, three,or more materials or excipients per phosphorylated hexaacyl disaccharidecontaining formulation described herein, and this limitation is notexclusive to only one material or excipient per phosphorylated hexaacyldisaccharide containing formulation. In another aspect, vaccines, andmethods of treating and preventing disease with vaccines, are provided.

To prepare the vaccines, one or more vaccine antigens are added to thephosphorylated hexaacyl disaccharide containing adjuvant formulationsdescribed above. The antigens may be a FimCH protein complex asdescribed herein or other antigens including, but not limited toantigens associated with diphtheria, tetanus, pertussis, poliomyelitis,hepatitis, and or antigenic preparations of the influenza virus.

A vaccine prepared using the phosphorylated hexaacyl disaccharideformulation does not require lyophilization to preserve theconcentration of phosphorylated hexaacyl disaccharide for shelf-life orstability. Lyophilization is a dehydration process or freeze-dryingprocess primarily utilized to preserve materials. Equivalent processesexist to accomplish the same goal to preserve materials. A vaccine oradjuvant formulation that does not need lyophilization is an unexpectedand significant advantage in the preparation of vaccines and thephosphorylated hexaacyl disaccharide compositions described herein. Byeliminating the need for lyophilization, many costly steps areeliminated. First, the lyophilization step itself is eliminated, whichnot only removes a costly manufacturing step that must occur underwell-monitored sterile conditions, but also eliminates the validationand review of this manufacturing step. Next, the removal of the steprepresents an ongoing cost saving. For example, each time a lot isprepared a lyophilization step is saved. In addition, removing this stepsaves extra validation steps when larger batches are prepared or themanufacturing procedure is transferred to another facility. Second,lyophilization requires that a sterile vial of diluent be manufacturedor procured, shipped, and stored with the lyophilized product forreconstitution. Eliminating the reconstitution step removes the cost ofthis diluent vial and its supply chain management. Third, thereconstitution of adjuvant formulations can compromise its sterility,necessitating its immediate use or waste of the product if not used in apre-established amount of time. Fourth, the reconstitution process isprone to errors, so the manufacturer loses control of the exactconcentration of the product that is ultimately administered to apatient. The adjuvant compositions and formulations described hereinhave eliminated these four disadvantages by removing the lyophilizationrequirement. Therefore, the phosphorylated hexaacyl disaccharidecontaining formulations are preferably not lyophilized or lyophilizationdoes not occur; the phosphorylated hexaacyl disaccharide containingformulations are preferably not dried after preparation; thephosphorylated hexaacyl disaccharide containing formulations preferablydo not require reconstitution from dried material with liquid or bufferafter preparation; and the phosphorylated hexaacyl disaccharidecontaining formulations preferably remain as an aqueous bufferedsuspension after manufacturing prior to administration.

A further remarkable advantage of the invention described herein is nowrealized. Without a need for lyophilization or equivalent process, thephosphorylated hexaacyl disaccharide compositions or phosphorylatedhexaacyl disaccharide containing formulations of the invention can beefficiently and cost effectively packaged into syringes immediatelyafter manufacturing. Preferably the compositions and formulations aresterile and preferably the syringes are sterile. These prefilledsyringes can be shipped, stored, delivered, or transferred atrefrigerated temperatures to room temperatures and up to about 37° C.This advantage provides the most efficient and cost effective means toget phosphorylated hexaacyl disaccharide compositions and phosphorylatedhexaacyl disaccharide containing formulations to a site foradministration.

In another embodiment, a non-ionic surfactant is added to the adjuvantformulation. Preferably the non-ionic surfactant is polysorbate 80although others may be used. The non-ionic surfactant is typically addedat a concentration of about 0.001% to 1.0%, preferably 0.01% to 0.1%, tothe adjuvant composition. This addition can prevent slight aggregationor slight increase in mean particle size of the phosphorylated hexaacyldisaccharide formulations while being stored at room temperatures and upto about 37° C. Preferably, non-ionic surfactants are derived frompolyethoxylated sorbitan and include but are not limited to polysorbate20 and polysorbate 80.

Preferred adjuvant formulations comprise a specific buffer selected fromthe group consisting of citrate, succinate, and phosphate at about 25 mMto about 50 mM, more preferably 28 mM to about 50 mM, and mostpreferably 30 mM to about 50 mM, and preferably one syntheticallyproduced phosphatidylcholine selected from the group consisting of DMPC,DPPC, DSPC, DOPC, and POPC, preferably DPPC, and one syntheticallyproduced adjuvant, phosphorylated hexaacyl disaccharide, in a molarratio of about 1:1 to 40:1 (phosphatidylcholine:phosphorylated hexaacyldisaccharide), preferably about 1:1 to 20:1(phosphatidylcholine:phosphorylated hexaacyl disaccharide), morepreferably about 2:1 to 5:1 (phosphatidylcholine:phosphorylated hexaacyldisaccharide), and most preferably about 2:1 to 5:1 (DPPC:phosphorylatedhexaacyl disaccharide). More preferably, citrate or succinate buffersare used in the phosphorylated hexaacyl disaccharide formulations atabout 10 mM to about 50 mM, preferably at about 25 mM to about 50 mM,more preferably 28 mM to about 50 mM, and most preferably 30 mM to about50 mM.

As described herein referring to the concentrations of the preferredbuffers of the phosphorylated hexaacyl disaccharide formulation, theconcentrations of the buffers are selected from the group consisting ofabout 10 mM to about 50 mM; 15 mM to about 50 mM; 20 mM to about 50 mM;about 25 mM to about 50 mM; 25 mM to about 50 mM; about 30 mM to about50 mM; 28 mM to about 50 mM; 30 mM to about 50 mM, about 30 mM; about 40mM to about 50 mM; 40 mM to about 50 mM; about 40 mM; and about 50 mM.The phosphorylated hexaacyl disaccharide containing formulationsdescribed herein are preferably substantially free of saline, preferablycontain less than 20 mM NaCl, and more preferably, the phosphorylatedhexaacyl disaccharide compositions contain less than 10 mM NaCl and evenmore preferably essentially free of NaCl.

In addition, as shown in the examples below, the use of PBS in thephosphorylated hexaacyl disaccharide containing compositions andformulations do not exhibit the novel characteristics of the invention,and in particularly the stability characteristics of the invention. Incontrast, the phosphate buffers as specifically defined herein do enablethe novel characteristics of the invention.

The phosphorylated hexaacyl disaccharide formulations are preferablyprepared by selecting a single phosphatidylcholine preparedsynthetically with high purity. However, naturally derivedphosphatidylcholines from soy or egg or hydrogenatedphosphatidylcholines from soy or egg or synthetic or natural mixed acylphosphatidylcholines may also be used to prepare the adjuvantformulations.

The phosphorylated hexaacyl disaccharide formulations can be prepared atany concentration but are typically prepared at about 0.005 to about 1.0mg/ml of phosphorylated hexaacyl disaccharide, preferably 0.05 to about1.0 mg/ml of phosphorylated hexaacyl disaccharide, but preferably notmore than about 2.5 mg/ml of phosphorylated hexaacyl disaccharide. Thephosphatidylcholine of the phosphorylated hexaacyl disaccharideformulation can be prepared at any concentration, but is preferablyprepared at about 0.005 to about 16 mg/mL (0.007 mM to 22 mM), morepreferably about 0.05 to about 8 mg/ml (0.07 mM to 11 mM), and even morepreferably about 0.05 to about 0.8 mg/ml (0.07 mM to 1 mM).

The phosphorylated hexaacyl disaccharide formulations of the inventionare prepared as follows to produce a molar ratio of about 40:1 to about1:1 of phosphatidylcholine to phosphorylated hexaacyl disaccharide,preferably DPPC to phosphorylated hexaacyl disaccharide, preferablyabout 2.5:1 DPPC to phosphorylated hexaacyl disaccharide. phosphorylatedhexaacyl disaccharide is weighed out into an appropriate glass vial,such as a Type 1 Plus Schott glass vial. An appropriate amount ofphosphatidylcholine, preferably DPPC, in ethanol is added. Thispreparation is sonicated for approximately 1 minute while gentlyswirling the preparation, and then the ethanol is appropriately removedvia evaporation. The film is reconstituted with citrate, succinate orphosphate buffer, preferably 10 mM to about 50 mM trisodium citrate, pH6.0, and sonicated at approximately 50° C. to 65° C., preferably 55° C.,for approximately 30 minutes, and more than one cycle of sonication canbe performed, as preferred. The prepared phosphorylated hexaacyldisaccharide formulation typically has particles sizes from about 60 nmto about 500 nm. To enable sterile filtration, the phosphorylatedhexaacyl disaccharide formulation is preferably further processed toachieve a reduced and appropriate homogenous particle size, preferablybetween about 70 nm to 130 nm. The phosphorylated hexaacyl disaccharideformulations are extruded through an 80 nm pore polycarbonate membrane(Avestin, LFLM-80) with an Avestin extruder, or equivalent, for about 7to about 12 passes at about 45° C. to 65° C., preferably 55° C. toachieve a homogenous particle size below about 130 nm. To ensure anacceptable recovery after sterile filtration, preferably the particlesizes of the phosphorylated hexaacyl disaccharide formulations are 150nm or less, but even more preferably 130 nm or less.

The phosphorylated hexaacyl disaccharide formulations may optionallythen be diluted with citrate, succinate or phosphate buffer atconcentrations described herein, but preferably at about 25 mM to about50 mM, more preferably 28 mM to about 50 mM, and most preferably 30 mMto about 50 mM, pH 6.0, containing an appropriate amount of polysorbate80 to achieve a final concentration of preferably 0.02% of polysorbate80, but 0.01% to 0.1% polysorbate 80 is acceptable. Typically, thephosphorylated hexaacyl disaccharide formulations will be diluted toabout a concentration of 0.05 mg/ml to 0.5 mg/mL. The phosphorylatedhexaacyl disaccharide formulations are then sterile filtered through a0.2 um filter, preferably Sartorius. The phosphorylated hexaacyldisaccharide and adjuvant formulations of the invention described hereinexhibit zeta potentials of about −20 mV to −80 mV.

The phosphorylated hexaacyl disaccharide formulations described herein,in one embodiment are used in the preparation of vaccines and areadministered to animals and humans as an adjuvant of vaccines.Preferably, the formulations are designed to deliver about 10 microgramsof phosphorylated hexaacyl disaccharide per dose to about 50 microgramsof phosphorylated hexaacyl disaccharide per dose, preferably about 20micrograms of phosphorylated hexaacyl disaccharide per dose to about 50micrograms of phosphorylated hexaacyl disaccharide per dose, and evenmore preferably about 40 micrograms of phosphorylated hexaacyldisaccharide per dose to about 50 micrograms of phosphorylated hexaacyldisaccharide per dose.

In a preferred embodiment, phosphorylated hexaacyl disaccharide isprovided as a single compound of approximately 98% purity with amolecular weight of 1763 Daltons (the structure of which is shown inFIG. 1. One source for the preferred phosphorylated hexaacyldisaccharide is Avanti Polar Lipids (Alabaster, Ala., USA) (PHAD). Thephosphorylated hexaacyl disaccharide compositions of the presentinvention, however, encompass phosphorylated hexaacyl disaccharide orpharmaceutically acceptable salts of phosphorylated hexaacyldisaccharide. The phosphorylated hexaacyl disaccharide used in thecompositions may be fully or partially synthetic or non-synthetic,although fully synthetic is preferred.

In other embodiments, derivatives of phosphorylated hexaacyldisaccharide, such as Monophosphoryl 3-Deacyl Lipid A (available fromAvanti Polar Lipids; referred to as 3D-PHAD) are used alone or incombination with phosphorylated hexaacyl disaccharide in thecompositions and formulations of the present invention.

PHAD's purity is in stark contrast to GSK's monophosphoryl lipid Aisolated from Salmonella minnesota that exists as a dynamic, complexmixture of hexa-, penta-, and tetraacyl analogues; each of theseanalogues differ in biological activity. Those skilled in the art ofdrug and vaccine development know that phosphorylated hexaacyldisaccharide is superior to GSK's monophosphoryl lipid A because PHAD'smanufacturing process, supply, use, and stability can be closelymonitored and controlled as a pure compound.

The concentration of citrate, succinate, or phosphate buffer is used toimprove stability of the phosphorylated hexaacyl disaccharideformulation at room temperature and up to about 37° C. While not boundby theory, it is believed that a synergistic effect results fromcomposition of molar ratio of PC:phosphorylated hexaacyl disaccharide,which is a preferred combination, and citrate, succinate, or phosphatebuffers within a specific concentration range as described herein.

Importantly, the phosphorylated hexaacyl disaccharide formulations arepreferably formulated to be substantially free of various excipients orchemicals. Therefore, the addition of cholesterol or two or morephosphatidylcholines or one or more phosphatidylglycerols is notrequired, and preferably not included in the formulations of theinvention. Adjuvant formulations essentially free of metabolizable oils,including squalene, and substantially free of cholesterol are preferred.In addition, while the prior art suggests cholesterol is a necessarychemical required for liposomes or adjuvant formulations, the adjuvantformulations described herein do not require cholesterol for theadjuvant to provide all the significant advantages over prior artformulations, and preferably no cholesterol is added to the adjuvantformulations. As shown in the Examples, two or more phosphatidylcholinesor one or more phosphatidylglycerols (for a total of two or morephosphatidylcholines or phosphatidylglycerols) may optionally be addedto the formulations at minor concentrations, but these are unnecessaryand not required to achieve stability at room temperature or up to about37° C. of the invention or to prevent, lower, or reduce severe injectionsite and systemic reactions while enhancing an immune response.

As detailed herein, those skilled in the art have been working withliposomes and other various formulations containing MLA, MPL, orsynthetic analogues of these adjuvants for more than twenty years andhave been unable to produce a formulation that allows for stability asdescribed herein while in aqueous suspensions. Room temperature and upto about 37° C. stability for vaccine adjuvants is a highly sought aftergoal by those skilled in the art. Despite the efforts, no formulationsto keep MLA, MPL, or synthetic analogues stable at the temperatures andfor the time periods described herein have been developed. The inventiondescribed herein solves this problem.

Preparation of Vaccines

Another embodiment of this invention further describes novel vaccinecompositions comprising the adjuvant or phosphorylated hexaacyldisaccharide formulations and FimCH or truncated FimH. Such vaccines areused to treat and prevent urinary tract infections caused bygram-negative bacteria including Escherichia coli and multi-drugresistant E. coli. FimCH is a non-covalent complex of FimC and FimHrecombinant proteins. A vaccine of FimCH and phosphorylated hexaacyldisaccharide formulation is prepared by adding a predetermined volume ofa phosphorylated hexaacyl disaccharide formulation to a vial of FimCH.

The following is an example of a vaccine prepared in accordance to theinvention. Generally, to prepare a vaccine for administration, aneffective amount of an antigen of FimCH or truncated FimH is combinedwith an adjuvant formulation containing about 0.005 mg/ml to about 0.5mg/ml of phosphorylated hexaacyl disaccharide to administer about 10 μgto about 50 μg of phosphorylated hexaacyl disaccharide per injection toa human.

In practice, the following is an example of one procedure that may beused by medical personnel to prepare and administer a vaccine inaccordance with the invention. The conditions and procedures areexemplary and do not limit the scope of the invention.

A vial of FimCH vial is removed from storage of about −20° C. andallowed to stand at room temperature for approximately twenty minutes toreach approximate room temperature. After the FimCH vial reachesapproximately room temperature, the vial is inverted a number of timesto mix the contents. Separately, a vial containing the phosphorylatedhexaacyl disaccharide formulation is removed from a storage containerfrom 2° C. to 8° C. storage. The vial of phosphorylated hexaacyldisaccharide formulation is inverted a number of times to mix thecontents, and then about 0.2 mL is withdrawn with a sterile 1.0 mLsyringe and injected into the FimCH vial through the stopper. Again thevial is inverted a number of times to mix the contents. Sterile waterfor injection (WFI) or more preferably preferred sterile buffer of theinvention is withdrawn in an amount of 0.2 mL using a sterile 1.0 mLsyringe and injected into the FimCH/phosphorylated hexaacyl disaccharidevial through the stopper. Again the vial is inverted a number of times.Finally, about 0.3 mL of prepared FimCH/phosphorylated hexaacyldisaccharide vaccine using a sterile 1.0 mL syringe is withdrawn. Theprepared vaccine contains 50 μg of FimCH and 20 μg of phosphorylatedhexaacyl disaccharide per a 0.3 mL dose. This prepared vaccine can bestored at refrigerated or room temperatures prior to administration.

In general FimCH is stored at −70° C., −20° C., or 2° C. to 8° C. forlong time periods, or even room temperature for a short time period ofabout 4 days to two to three weeks. Typically, only sterile productsshould be stored at room temperature because if the products are notsterile, microbial growth is possible, but not guaranteed. The vaccineis preferably administered by intramuscular injection. Typically, about5 micrograms of FimCH to about 200 micrograms of FimCH would beadministered to a human, preferably about 20 micrograms to about 110micrograms. Typically, about 10 micrograms of phosphorylated hexaacyldisaccharide per dose to about 50 micrograms of phosphorylated hexaacyldisaccharide per dose would be administered with FimCH, more preferablyabout 20 micrograms of phosphorylated hexaacyl disaccharide per dose toabout 50 micrograms of phosphorylated hexaacyl disaccharide per dose,even more preferably about 40 micrograms of phosphorylated hexaacyldisaccharide per dose to about 50 micrograms of phosphorylated hexaacyldisaccharide per dose although more or less may be used. Typically,three to four doses of FimCH with the phosphorylated hexaacyldisaccharide formulation is administered to a patient in need. Thesedoses typically occur at day 0 and then about days 30 to 60, then about90 to 180 days, and then, if preferred, about 180 to 360 days from thefirst administration. As needed, additional injections may occur 12 to36 months after the initial vaccination.

As described above, the more preferable aspect of the invention is thatthe phosphorylated hexaacyl disaccharide compositions and formulationsare stored separately from the antigen or FimCH because thephosphorylated hexaacyl disaccharide compositions and formulations haveexcellent stability, and formulated or mixed appropriately with theantigen or FimCH sometime before administration to a patient or human.However, other methods of mixing, preparing and administering vaccinesare possible and will function effectively.

The data in the following sections demonstrates that the adjuvantformulations of the invention enhance the immune response of otherantigens including bacterial and viral antigens. One or more vaccineantigens may be added to the phosphorylated hexaacyl disaccharideformulations prepared in accordance with the invention. These antigensmay be the FimCH protein complex as described herein or other antigensincluding, but not limited to diphtheria, tetanus, pertussis,poliomyelitis, hepatitis, and or antigenic preparations of the influenzavirus.

In another embodiment, methods of administration of the novel vaccinecompositions comprising adjuvant or phosphorylated hexaacyl disaccharideformulations and FimCH or truncated FimH are provided. Particularly,methods of treatment to prevent and treat urinary tract infectionscaused by gram-negative bacteria including E. coli and multi-drugresistant E. coli are provided. Typically, about 5 micrograms of FimCHto about 200 micrograms of FimCH would be administered to a human,preferably about 20 micrograms to about 110 micrograms. Typically, about10 micrograms of phosphorylated hexaacyl disaccharide per dose to about50 micrograms of phosphorylated hexaacyl disaccharide per dose would beadministered, preferably about 20 micrograms of phosphorylated hexaacyldisaccharide per dose to about 50 micrograms of phosphorylated hexaacyldisaccharide per dose, even more preferably about 40 micrograms ofphosphorylated hexaacyl disaccharide per dose to about 50 micrograms ofphosphorylated hexaacyl disaccharide per dose. Other dosage amounts andregimens may be used dependent on the antigen used and the conditionbeing treated.

In another embodiment, methods of inducing the production of antibodiesagainst FimH in a human with recurrent urinary tract infections areprovided.

In another embodiment, vaccine compositions that induce the productionof antibodies against FimH in a human with recurrent urinary tractinfections are provided.

In another embodiment, sterile compositions and sterile pharmaceuticalcompositions containing phosphorylated hexaacyl disaccharide areprovided; preferably these compositions of aqueous buffered suspensionshave particle sizes less than 150 nm, even more preferably less than 130nm. The sterile phosphorylated hexaacyl disaccharide compositions andformulations are stored in a pharmaceutical container which is in directcontact to the phosphorylated hexaacyl disaccharide compositions andformulations. Examples of these pharmaceutical containers are vials orsyringes, more preferably the sterile phosphorylated hexaacyldisaccharide compositions and formulations are contained in a sterilesyringe. These pharmaceutical containers holding the phosphorylatedhexaacyl disaccharide composition or formulation can be stored in atemperature-validated container, e.g. incubator, at room temperature.These pharmaceutical containers holding the sterile phosphorylatedhexaacyl disaccharide composition formulation can be added to a shippingcontainer assembled to be transferred to another location at roomtemperature or up to about 37° C. These shipping containers can betransferred via a government postal service or a commercial shippingservice. Due to the remarkable stability of the inventions describedherein, the location receiving said phosphorylated hexaacyl disaccharidecompositions and formulations may be a location without refrigeration orintermittent access to refrigeration or a location without electricityor with intermittent electricity.

In another embodiment, a vaccine kit comprising the phosphorylatedhexaacyl disaccharide or adjuvant compositions or formulations orvaccine composition is provided. The kit may optionally include methodsof preparing and administering the vaccine and/or instructions forstorage and exposure of the phosphorylated hexaacyl disaccharidecompositions or formulations at room temperature and up to and about 37°C. These instructions describing storage, shipping, and exposuretemperatures may be approved by a government regulatory authorityincluding the US FDA or European Medicines Agency. Preferably, one ormore of the kit components is the phosphorylated hexaacyl disaccharidecompositions or formulations in a syringe. Preferably the phosphorylatedhexaacyl disaccharide compositions or phosphorylated hexaacyldisaccharide containing formulations are sterile and are in a sterilesyringe.

The kit may include a label for the phosphorylated hexaacyl disaccharideor adjuvant compositions or formulations of the invention providinginstructions or limitations for storage and exposure of thephosphorylated hexaacyl disaccharide compositions or formulations atroom temperature and up to and about 37° C. The labels or instructionsdescribing storage, shipping, and exposure temperatures may be approvedby a government regulatory authority including the US FDA or EuropeanMedicines Agency.

As stated herein, the novel characteristics of the invention enable thephosphorylated hexaacyl disaccharide compositions and formulations to bemanufactured, tested, analyzed, stored, shipped, held, moved,transferred, or administered at refrigerated temperatures, roomtemperature, up to and about 37° C., temperatures between refrigeratedtemperatures and room temperature for periods of time as describedherein. Due to the remarkable stability of the inventions describedherein, the location receiving said phosphorylated hexaacyl disaccharidecompositions and formulations may be a location without refrigeration orintermittent access to refrigeration or a location without electricityor with intermittent electricity.

EXAMPLES

Certain specific aspects and embodiments of the present disclosure willbe explained in more detail with reference to the following examples,which are provided solely for purposes of illustration and are not to beconstrued as limiting the scope of the disclosure in any manner. Theamounts used do not represent a limitation and the process can be scaledup to produce larger batches

Example 1

Phosphorylated hexaacyl disaccharide formulations are manufactured asfollows to produce a molar ratio of about 2.5:1 of DPPC tophosphorylated hexaacyl disaccharide.

PHAD Avanti Polar Lipids (Alabaster, Ala., USA), phosphorylated hexaacyldisaccharide, and DPPC can be obtained from Avanti Polar Lipids(Alabaster, Ala., USA) as either non-GMP or GMP material (theCertificate of Analysis is provided in Table 1). phosphorylated hexaacyldisaccharide is a synthetic version of monophosphoryl lipid A.phosphorylated hexaacyl disaccharide is formulated with DPPC to preparethe adjuvant formulation of the invention. DPPC's release specificationsare provided in the table 2. The transition temperature of DPPC is 41°C.

PHAD is weighed out into an appropriate glass vial, preferably a Type 1Plus Schott glass vial. An appropriate amount of an approximately 2.3mg/ml DPPC solution, or equivalent, in ethanol is added. Thispreparation is sonicated for approximately 1 minute while gentlyswirling the preparation, and then the ethanol is appropriately removedvia evaporation using care. The film is reconstituted with 10 mMtrisodium citrate, pH 6.0, and sonicated at approximately 50° C. to 65°C., preferably 55° C., for approximately 30 minutes. The phosphorylatedhexaacyl disaccharide formulations are typically prepared at about 0.5to 1.0 mg/ml, but not more than about 2.5 mg/ml. (As described herein,molar ratios of for example about 13:1 DPPC:phosphorylated hexaacyldisaccharide can also be prepared using this same procedure by adjustingthe amount of DPPC as needed.

The prepared PHAD formulations typically exhibit particles sizes fromabout 60 nm to about 500 nm. To enable sterile filtration, thephosphorylated hexaacyl disaccharide formulations have to be furtherprocessed to achieve a reduced and appropriate homogenous particle size,typically between about 70 nm to 130 nm. Even though numerous literaturereferences, including U.S. Pat. No. 6,630,161, report high-pressurehomogenization is a preferred method to reduce the particle size ofliposomes, high-pressure homogenization using an Avestin homogenizer ofpressures up to 25,000 psi do not significantly or relevantly reduce theparticle sizes of these phosphorylated hexaacyl disaccharideformulations. This difficulty was unexpected. These phosphorylatedhexaacyl disaccharide formulations must be extruded through an 80 nmpore polycarbonate membrane (Avestin, LFLM-80) with an Avestin extruder,or equivalent, for about 7 to about 12 passes at about 45° C. to 65° C.,preferably 55° C. to achieve a homogenous particle size below about 130nm. Extruding at 45° C. to 65° C. is an important parameter. To ensurean acceptable recovery after sterile filtration, the particle sizes ofthe phosphorylated hexaacyl disaccharide adjuvant formulations arepreferably 150 nm or less, but more preferably 130 nm or less.

The phosphorylated hexaacyl disaccharide formulations may then bediluted with 10 mM trisodium citrate, pH 6.0 containing an appropriateamount of polysorbate 80 to achieve a final concentration of mostpreferably 0.02% of polysorbate 80, but 0.01% to 0.1% polysorbate 80 isacceptable. Typically phosphorylated hexaacyl disaccharide formulationswill be diluted to about a concentration of 0.05 mg/ml to 0.5 mg/mL.These phosphorylated hexaacyl disaccharide formulations are then sterilefiltered through a 0.2 um filter, preferably Sartorius.

As determine by cryogenic transmission electron microscopy, the adjuvantformulations described herein are suspensions.

Additional or alternative steps may be added to the procedure above toprepare the phosphorylated hexaacyl disaccharide formulations. For oneexample, the ethanol may be evaporated by rotary evaporation, orequivalent, or via a nitrogen stream, or equivalent. For anotherexample, the sonication step including a buffer of the invention may berepeated two or more times, the formulation may be cooled to roomtemperature or less between repeated sonications, and the formulationmay be held at a temperature similar to the sonication step for one ormore hours before, during, or after said sonication.

TABLE 1 Certificate of Analysis Information for PHAD from Avanti PolarLipids, Inc. Analysis Specification Results Physical examination Whiteto off-white powder Pass or lyophilized cake which contains no foreignmatter. TLC Ninhydrin spray, negative All pass (65:25:4 (v/v/v) Iodine,1 major spot chloroform: Phosphorus spray, positive methanol:water)Charring, positive Water dip, 1 major spot Rf consistent with structureHPLC NLT 97% purity 99.2% Proton NMR Consistent with structureConsistent with structure Phosphorus NMR Consistent with structureConsistent with structure MS Consistent with structure Consistent with(exact mass = 1762.3) structure Karl Fischer NMT 5% water  1.4% WaterTitration Residual Solvents NMT 2000 ppm methanol None Detected(GC/FID)* NMT 2000 ppm ethanol None Detected NMT 2000 ppm acetone NoneDetected NMT 200 ppm hexane None Detected NMT 2000 ppm None Detectedcyclohexane None Detected NMT 500 ppm toluene None Detected NMT 50 ppmNone Detected chloroform/ethyl acetate NMT 5000 ppm total residualsolvents Palladium (ICPMS)* NMT 10 ppm <0.1 ppm Heavy Metals Screen +NMT 20 ppm  <20 ppm Ruthenium + Iridium by ICPMS*

TABLE 2 Certificate of Analysis Information for DPPC from Avanti PolarLipids, Inc. Analysis Specification Results Physical examination Whitesolid which contains Pass no foreign matter. TLC Ninhydrin spray,negative All Pass Iodine, 1 major spot Phosphorus spray, positiveCharring, negative Water dip, 1 major spot Rf consistent with structureQuantitative NMT 1% 16:0 dimethyl PE None detected Phosphorus NMR HPLCNMT 1% palmitic acid None detected NMT 1% 16:0 lyso PC  0.7% NLT 99%purity  99.3% Fatty acid methyl ester NLT 99% (AUC) palmitoyl 100.0% byGC/FID methyl ester Karl Fischer Water NMT 8% water  0.5% TitrationResidual Solvents by NMT 100 ppm methanol None Detected GC/FID NMT 100ppm ethanol None Detected NMT 100 ppm acetone None Detected NMT 100 ppmhexane None Detected NMT 100 ppm cyclohexane None Detected NMT 100 ppmtoluene None Detected NMT 20 ppm chloroform None Detected NMT 250 ppmtotal residual None Detected solvents

Example 2

Antigens for the vaccine may be prepared as follows.

FimCH is a non-covalent complex of FimC and FimH recombinant proteins.The recombinant proteins are derived from transgenic E. coli culture.The FimC and FimH proteins are expressed separately in E. coli, and theyspontaneously form a non-covalent complex. The molecular weight of theFimCH complex is approximately 51,700 Daltons.

The FimH protein (SEQ ID No: 1) of the complex has a molecular weight of29,065 Daltons, and it consists of 279 amino acid residues representedby the sequence below:

Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly Ser AlaAsn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln Asn Leu Val ValAsp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr Pro Glu Thr Ile Thr AspTyr Val Thr Leu Gln Arg Gly Ser Ala Tyr Gly Gly Val Leu Ser Asn Phe SerGly Thr Val Lys Tyr Ser Gly Ser Ser Tyr Pro Phe Pro Thr Thr Ser Glu ThrPro Arg Val Val Tyr Asn Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu TyrLeu Thr Pro Val Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu IleAla Val Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln PheVal Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly Cys AspVal Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Arg Gly Ser Val ProIle Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn Leu Gly Tyr Tyr Leu SerGly Thr His Ala Asp Ala Gly Asn Ser Ile Phe Thr Asn Thr Ala Ser Phe SerPro Ala Gln Gly Val Gly Val Gln Leu Thr Arg Asn Gly Thr Ile Ile Pro AlaAsn Asn Thr Val Ser Leu Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly LeuThr Ala Asn Tyr Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln SerIle Ile Gly Val Thr Phe Val Tyr Gln

The FimC (SEQ ID No: 2) protein of the complex has a molecular weight of22,700 Daltons, and it consists of 205 amino acid residues representedby the following sequence:

Gly Val Ala Leu Gly Ala Thr Arg Val Ile Tyr Pro Ala Gly Gln Lys Gln ValGln Leu Ala Val Thr Asn Asn Asp Glu Asn Ser Thr Tyr Leu Ile Gln Ser TrpVal Glu Asn Ala Asp Gly Val Lys Asp Gly Arg Phe Ile Val Thr Pro Pro LeuPhe Ala Met Lys Gly Lys Lys Glu Asn Thr Leu Arg Ile Leu Asp Ala Thr AsnAsn Gln Leu Pro Gln Asp Arg Glu Ser Leu Phe Trp Met Asn Val Lys Ala IlePro Ser Met Asp Lys Ser Lys Leu Thr Glu Asn Thr Leu Gln Leu Ala Ile IleSer Arg Ile Lys Leu Tyr Tyr Arg Pro Ala Lys Leu Ala Leu Pro Pro Asp GlnAla Ala Glu Lys Leu Arg Phe Arg Arg Ser Ala Asn Ser Leu Thr Leu Ile AsnPro Thr Pro Tyr Tyr Leu Thr Val Thr Glu Leu Asn Ala Gly Thr Arg Val LeuGlu Asn Ala Leu Val Pro Pro Met Gly Glu Ser Ala Val Lys Leu Pro Ser AspAla Gly Ser Asn Ile Thr Tyr Arg Thr Ile Asn Asp Tyr Gly Ala Leu Thr ProLys Met Thr Gly Val Met Glu

For producing the transgenic cell line, the FimC gene from E. colistrain J96 was amplified with primers SLC4-28-fimC5 and SLC4-28-FimC3from purified J96 genomic DNA to give a 771 base pair product. This wasdigested with BamHI and EcoRI, purified, and ligated into pTRC99a cutwith the same enzymes (BamHI and EcoRI). The ligation product wastransformed into E. coli C600 cells, and selected on ampicillin,producing plasmid pSJH-32.

The ampicillin antibiotic resistance was switched to kanamycin using thefollowing procedure: Primers pKD4-pr1 and pKD4-pr2 were used to amplifythe kanamycin resistance gene from pKD4. This PCR product wasphosphorylated with T4 polynucleotide kinase and gel purified. pSJH-32was cut with ScaI and BglI, blunted with T4 DNA polymerase,dephosphorylated with calf intestinal alkaline phosphatase, then ligatedwith the phosphorylated kanamycin resistance gene PCR product. Theligation product was then transformed into E. coli C600 cells andselected on kanamycin, creating plasmid pSJH-319.

The FimH gene from strain J96 was amplified with primers FimH5 and FimH3from purified J96 genomic DNA to give a 978 base pair product. It wasdigested with SacI and HindIII, purified, and ligated into pBAD33digested with SacI and HindIII. Afterwards the construct was transformedinto C600 cells, and selected on chloramphenicol.

Example 3

Bioprocessing (Process to obtain antigens of the vaccine).

The bioprocessing step is initiated with inoculation of master cell bank(MCB) into shake flasks containing APS LB medium with kanamycin (50μg/mL) and chloramphenicol (20 μg/mL). When the OD reaches 2.0-3.0 units(after approximately 15 hours growth), the cell culture is transferredaseptically into reactors for fed-batch fermentation. The mediumcontaining APS Super Broth, about 0.8% glycerol, and antibiotics issterilized prior to inoculation. FimH protein expression is induced atOD 10 with IPTG. Five minutes after IPTG addition, FimC proteinexpression is induced with arabinose. Cells are harvested approximatelyone hour later. After harvesting, the cells are separated from the mediacomponents by continuous or batch centrifugation.

Protein Recovery

Recombinant FimCH is expressed in the E. coli periplasm. E. coli, as agram negative bacteria, possess an inner and an outer lipid bilayermembrane. The space between the lipid bilayers is the periplasm.Immediately after centrifugation, FimCH is recovered from the cell usinga periplasm preparation. The cells are reacted with recombinant lysozymein the presence of sucrose, Tris, and EDTA at 2-8° C. The mixture isthen centrifuged, and the resulting periplasmic protein solution iscollected. The protein is then precipitated with ammonium sulfate,centrifuged, resuspended in 20 mM MES pH 5.9, and diafiltered viadialysis into 20 mM MES pH 5.9 using SpectraPor 2 Dialysis Membrane(Spectrum Labs 132680). When the solution conductivity decreases toapproximately ≦1.5 mS/cm, the solution is collected and transferred topurification.

Protein Purification

Purification consists of three column chromatography steps (1.CEX, 2.HIC, 3. CEX), one buffer exchange step via diafiltration usingSpectraPor 2 Dialysis Membrane (Spectrum Labs 132680) followed byfiltration, and one final aseptic filtration step. The diafiltrationstep is used to exchange the protein into 20 mM MES buffer pH 5.9 sothat it will bind the second CEX column.

The two CEX steps use Source 15S (GE Healthcare 17-1273-02) in an XK26column. For both CEX columns the following conditions are used: BufferA: 20 mM MES, pH 5.9; Buffer B: 20 mM MES/500 mM Sodium Chloride, pH5.9; 8 ml/min for all steps except loading for XK26/10 column,pre-equilibrate the column with 5 CV of Buffer B, equilibrate the columnwith 4 CV of Buffer A, load Dialyzed FimCH sample at 5 ml/min forXK26/10 using a sample pump and specifically not via the chromatographypump, wash the column with 4 CV of Buffer A, and elute the column with alinear 5 CV gradient from 0-25% Buffer B with fraction collection.

For the HIC column used Butyl Sepharose 4FF (GE Healthcare 17-0980-01)in XK26 column. Buffer C: 20 mM MES/550 mM Ammonium Sulfate, pH 5.9; 8ml/min for XK26/10 except loading, pre-equilibrate the column with 3 CVof Buffer A (as above), equilibrate the column with 6 CV of Buffer C,load Pooled FimCH sample at 5 ml/min XK26/10 column, wash the columnwith 6 CV of Buffer C, elute the column with a linear 4 CV gradient from0-100% Buffer A with fraction collection. FimCH is formulated in theconcentration of 0.3 mg/mL in 20 mM MES pH 5.9 or 20 mM trisodiumcitrate, pH 5.4. It is then aseptically filtered through a 0.2 μmsterile filter. FimCH is stable and can be stored at −20° C. for aboutat least 2 years.

Example 4

Potency by In Vitro Mannose Binding (Demonstrates the biologicalactivity of FimCH).

The biological activity of the FimCH drug substance (for example, fromExample 3) is determined by an in vitro mannose binding assay. The FimHprotein is a bacterial adhesin utilized by E. coli to bind mannoseresidues on glycosylated proteins. During urinary tract infections, theFimH adhesin binds mannosylated uroplakin proteins on bladder epithelialcells, which promotes the internalization of bound E. coli. The bindingof FimH to mannosylated uroplakin is essential for E. coli to causeurinary tract infections. To monitor the mannose-binding activity ofFimH in vitro, FimH binding to the enzyme horseradish peroxidase (HRP)is observed. HRP is a glycosylated protein containing mannose residues,and has previously been used to study mammalian mannose-bindingreceptors. Complexes of HRP and the lectin ConA, which bindsα-D-mannosyl and α-D-glucosyl groups, have also been generated andstudied. These results demonstrate that HRP acts as a ligand for otherknown mannose-binding proteins. Using this potency assay as describedbelow, HRP binding to FimH is shown to be concentration-dependent and tobe inhibited by small molecules that the block the binding of mannose toFimH.

In the in vitro FimCH potency assay, FimCH is “captured” by purified andqualified anti-FimH antisera bound to an ELISA plate. The anti-FimHantisera used in this assay have demonstrated the ability to bind toFimH in both indirect ELISAs (as the detection antisera, FIG. 3) andwestern blots. HRP is then added, excess HRP is rinsed away, and theactivity of bound HRP is detected. The measured HRP activity isproportional to the concentration of FimCH added (FIG. 4). These resultsdemonstrate that HRP binds to FimH in a dose-dependent manner.

To demonstrate that HRP binding to FimH requires FimH mannose-bindingactivity, a mannose binding-deficient FimH mutant named Q133K, which isalso in complex with FimC, was analyzed and compared to FimCH. Q133Kshares the same amino acid sequence as FimH, except that a criticalglutamine at position 133 is replaced by lysine. This mutation is in theFimH mannose-binding pocket and renders Q133K functionally deficient inbinding mannose and mannosylated proteins. As shown in FIG. 4, Q133KFimCH does not bind HRP. In an indirect ELISA (FIG. 3), the Q133K mutantcomplex is recognized by the purified anti-FimH antisera. Thisdemonstrates that the lack of HRP signal with Q133K is not due to aninability of the purified antisera to bind to Q133K; instead, it is dueto the inability of Q133K to bind mannose residues on HRP. These resultsalso demonstrate that HRP does not bind FimC, because Q133K is also incomplex with FimC.

As demonstrated in Hung et al. 2002, single point mutations in FimH atpositions 54, 133, 135, and 140 completely abolish mannose binding. Asreported by Hung et al., “ . . . even the slightest change in themannose-binding pocket, in an atom that does not bind directly tomannose, significantly reduces binding,” suggesting that mutations thatcould occur in vitro could severely limit or abolish FimH mannosebinding activity. The lack of HRP binding to the Q133K mutant supportsthis assay's ability to assess the biological activity of FimH bindingto mannosylated proteins.

Several small molecule inhibitors of mannose binding to FimH have beendescribed. Two of these inhibitors,4-methylumbelliferyl-α-D-mannopyranoside (UFMP) andmethyl-α-D-mannopyranoside (MDMP), have been used to further qualifythis potency assay. The reported dissociation constant (Kd) for UFMPbinding to FimH is 20 nM, which is approximately 100-fold more potentthan MDMP's Kd of 2.2 μM. As expected, the addition of either of theseFimH mannose binding inhibitors in the HRP binding step blocks HRPbinding to FimH in a dose-dependent manner (see FIG. 5). For UFMP, 50%inhibition is observed at 10 ng/mL (30 nM). For MDMP, 50% inhibition isobserved at approximately 1 μg/mL (5.1 μM), which is approximately100-fold higher than the concentration of UFMP.

These results demonstrate this assay's ability to assess the biologicalactivity of FimH and verify the consistency of the manufacturing processfrom batch to batch. Furthermore, this potency assay confirms the properfolding of the FimH epitope and is predictive of the generation of IgGanti-FimH that has been shown to reduce E. coli CFU in bladders of miceby the administration of the FimCH/phosphorylated hexaacyl disaccharidevaccine.

Example 5 Impurities of FimCH Drug Substance by CEX-HPLC

CEX-HPLC is used for determination of the FimCH complex, unbound FimCand impurities in the final FimCH drug substance. The protein is elutedfrom a GE Healthcare Mono S 5/50 GL column using a gradient of 0.3 MNaCl in 20 mM MES buffer, pH 6.2 (Buffer B) (Buffer A is 20 mM MESbuffer, pH 6.2). At T=0 the mobile phase is 100% Buffer A, and at T=22minutes, the mobile phase is 100% Buffer B. The relative content of theunbound FimC and impurities is determined based on the peak areas. Arepresentative chromatogram is provided in FIG. 6.

Example 6

FimCH and phosphorylated hexaacyl disaccharide formulation: An 85-DayIntramuscular Toxicity/Immunogenicity Study in Rabbits with a 21 DayRecovery Period (GLP).

The pivotal GLP toxicity study to evaluate the toxicity andimmunogenicity of the FimCH vaccine containing a phosphorylated hexaacyldisaccharide formulation of the invention (DPPC:PHAD—about 2.4:1 molarratio) was conducted in female rabbits. The study examinedophthalmological findings, antibody assessment, and histopathology.Female rabbits were administered a total of 5 doses of saline control(N=6), phosphorylated hexaacyl disaccharide alone at 40 to 50 μg (N=12),FimCH at 100 μg plus 20 μg phosphorylated hexaacyl disaccharide (N=6;low dose), or FimCH at 125 μg plus 40 to 50 μg phosphorylated hexaacyldisaccharide (N=12; high dose) via IM injection every 3 weeks (Days 1,22, 43, 64, and 85) for 13 weeks. Three days following the fifth dose(Day 88), 6 rabbits per group were euthanized with the remaining 6rabbits in the phosphorylated hexaacyl disaccharide alone group andFimCH high dose groups euthanized following a 3-week recovery period(Day 106).

Toxicity was assessed based on clinical observation, ophthalmology, bodytemperature, body weight, food consumption, clinical pathology, grossnecropsy, organ weight, and histopathology data. Body temperatures wereobtained prior to and 2, 4, 6, 24, 48 and 72 hours after each injection.In addition to standard clinical pathology parameters (predose, Days 2and 88), C-reactive protein (CRP) and fibrinogen were evaluated 2 and 7days after dosing. Potential injection site reactions were scored foredema and erythema using the Draize scale and assessed for othermanifestations of local toxicity (i.e. eschar, vesiculation, ulceration,and hematoma) 24, 48, and 72 hours post dose. Anti-FimH antibodyassessment was performed on serum samples collected prior dosing on Days1, 22, 43, 64 and 85, and prior to necropsy on Days 88 and 106, on urinesamples collected prior to dosing, Day 64, and prior to necropsy on Days88 and 106, and on vaginal washings collected prior dosing on Days 1and, and prior to necropsy on Days 88 and 106. Antibody evaluation ofthe urine and vaginal wash samples was qualitative using a qualifiedMSD-ECL assay. Antibody levels in serum were determined using avalidated MSD-ECL assay. Of 18 rabbits vaccinated with FimCH andphosphorylated hexaacyl disaccharide, 17 demonstrated anti-FimH IgGtiters at about 1:3,200,000 at about Day 88.

All rabbits survived to scheduled necropsy. Preliminary data indicatedthat FimCH plus phosphorylated hexaacyl disaccharide as well asphosphorylated hexaacyl disaccharide alone were well tolerated. Thefindings demonstrated that there were no apparent phosphorylatedhexaacyl disaccharide alone or vaccine-related effects on clinicalobservations, body weight, food consumption, body temperature, clinicalpathology, or organ weights. Local reaction, based on in-lifeobservations, was limited.

A similar rabbit study was performed using FimCH with PHAD prepared asDPPC:phosphorylated hexaacyl disaccharide at about a molar ratio of1:3.9. In this study, only 5 of 16 rabbits at about Day 43 demonstratedanti-FimH IgG titers from 1:400,000 to 1:800,000. In contrast, 16 of 18rabbits vaccinated with FimCH with phosphorylated hexaacyl disaccharideprepared as DPPC:phosphorylated hexaacyl disaccharide at about a molarratio of 2.4:1 (listed above) demonstrated anti-FimH IgG titers from1:400,000 to 3,200,000 at about Day 43. These immunogenic differencesare consistent with the studies performed in mice described hereindemonstrating that a molar ratio of DPPC:phosphorylated hexaacyldisaccharide of about 2.4:1 is superior to a molar ratio ofDPPC:phosphorylated hexaacyl disaccharide of about 1:3.9.

Example 7

To test the efficacy of phosphorylated hexaacyl disaccharide as anadjuvant for systemic vaccination in the mouse UTI infection model,C3H/HeN mice were infected with approximately 1×10⁸ CFU of clinicalcystitis E. coli isolate UTI89 via transurethral catheterization afterIM immunization. Female C3H/HeN mice (approximately 9 weeks old) werepurchased from Charles River laboratories (Wilmington, Mass.). Mice wereinjected via the intramuscular route (IM) in the right thigh under lightisoflurane anesthesia (Henry Schein, Melville, N.Y.) in a 50 μl volumeusing a 30 gauge needle. In these efficacy studies, mice were immunizedwith 12.5 μg PHAD/15 μg FimCH. Mice immunized using phosphorylatedhexaacyl disaccharide as the adjuvant and FimCH as antigen showed astatistically significant decrease of E. coli CFU in bladders one or twodays after infection compared to mice immunized with adjuvant alone andto naïve mice (experiment using PHAD shown in FIG. 7). These datademonstrate that a FimCH vaccine adjuvanted with phosphorylated hexaacyldisaccharide produces antibodies in mice that reduce the E. colicolonization of bladders. These data offer evidence that a FimCH vaccineadjuvanted with phosphorylated hexaacyl disaccharide prepared accordingto the compositions described herein and used according to the methodsdescribed herein administered to a human patient in need will alsoreduce the E. coli colonization of bladders in humans.

Example 8

Truncated FimH (FimHt) adjuvanted with phosphorylated hexaacyldisaccharide formulation or Freund's adjuvant: An Immunogenicity Studyin Rabbits.

Female rabbits were administered a total of 3 doses of FimHt at 100 μgplus about 50 μg phosphorylated hexaacyl disaccharide formulation of theinvention (N=2) on days 0, 21, and 42, or a total of 5 doses of FimHt at100 μg plus Freund's adjuvant (complete for initial vaccination andincomplete for each boost at about days 14, 21, 49, and 70) (N=2) via IMinjection. In this experiment, truncated FimH has a series of histidinesor histidine tag, and those skilled in the art understand that othertruncated versions of FimH can also be used, most preferably requiringthe mannose binding domain. Truncated FimH in this example consists ofFimH residues 1 to 175 with a C-terminal 6-histidine tag (SEQ ID No: 3).The sequence of FimH is described in Example 2. Anti-FimH antibodyassessments were performed on serum samples collected on about day 30 orabout days 35 and 56. Antibody levels in serum were determined using anELISA as described herein. The capture antigen for this experiment wasan equivalent truncated FimH without a histidine tag. Rabbits vaccinatedwith both formulations demonstrated anti-FimH IgG greater than1:1,600,000 (preimmune sera <1:10,000). Previous truncated versions ofFimH have been publicly disclosed and one example is as in U.S. Pat. No.6,737,063, which is specifically incorporated in its entirety.

Example 9 FimCH Vaccine with phosphorylated hexaacyl disaccharideFormulation Administered to Rabbits

Two groups of rabbits (N=3) were immunized at day 0 and boosted at days21, 42, via IM injection with 50 μg FimCH and 54 μg phosphorylatedhexaacyl disaccharide with and without 0.1% polysorbate 80(phosphorylated hexaacyl disaccharide formulation of the invention, 10mM trisodium citrate, pH 6.0). Antibody levels in serum were determinedusing an ELISA as described herein. Serum was collected from both groupsincluding at about day 30. IgG anti-FimH titers were approximatelyequivalent to or more than 1:1,600,000 in both groups (preimmune sera<1:10,000). The data described herein demonstrate that FimCH vaccinewith phosphorylated hexaacyl disaccharide formulations of the inventionwith or without polysorbate 80 generate an equivalent immunogenicresponse.

Example 10 HPLC Analysis of phosphorylated hexaacyl disaccharide andDPPC in Compositions

phosphorylated hexaacyl disaccharide and DPPC concentrations in thephosphorylated hexaacyl disaccharide formulation are analyzed byHPLC-ELSD using an Agilent Eclipse XBD C18, 1.8 um, 4.6 mm×50 mm column.The mobile phases are as follows: MP A: 20 mM ammonium acetate/1% aceticacid in water; MP B: 20 mM ammonium acetate/1% acetic acid in methanol;and MP C: 20 mM ammonium acetate/1% acetic acid in methanol/chloroform(50/50). Method 1: gradient begins at 5% MP A and 95% MP B, at 2 minutesis 100% MP B, and at 8 minutes is 100% MP C. Method 2:gradient begins at5% MP A and 95% MP B, at 2 minutes is 100% MP B, and at 15 minutes is100% MP C. Sample Diluent 1: 85:15 (75:15:10 methanol:chloroform:waterwith 20 mM ammonium acetate/1% acetic acid):(1:1 methanol:chloroformwith 20 mM ammonium acetate/1% acetic acid) Sample and standards arediluted 1:4 with sample diluent 1. Sample Diluent 2: (70:25:5)methanol:chloroform:water with 20 mM ammonium acetate/1% acetic acid. Ifusing sample diluent 2, sample and standards are diluted 1:10 withsample diluent 2. The ELSD gain is at 8 with temperature at 60 C andnitrogen flow set to approximately 3.7 bars. An example chromatogramusing method 2 and sample diluent 2 is shown in FIG. 8.

Example 11

Stability studies of phosphorylated hexaacyl disaccharide compositionsand formulations.

Using the HPLC method as described in Example 10, the stabilities ofdifferent preparations of phosphorylated hexaacyl disaccharide as asuspension or phosphorylated hexaacyl disaccharide formulations as asuspension including phosphatidylcholines were monitored by analyzingthese preparations for phosphorylated hexaacyl disaccharideconcentration and comparing it to its initial results. A PASS resultmeans the phosphorylated hexaacyl disaccharide concentration was withinplus/minus 20% of the initial testing results of the release samples,which is within the limits of the HPLC method using an evaporative lightscattering detector (ELSD). phosphorylated hexaacyl disaccharideconcentrations of these preparations were compared when stored at 2° C.to 8° C. or 25° C. or to 37° C. to project (estimate) the stability ofthese preparation over months to years. To those skilled in the art,data from 25° C. is an intermediate/accelerated condition and data from37° C. is an accelerated condition used to project a shelf life at thelong term storage condition of 2° C. to 8° C. Buffers that enabledsuperior stability for phosphorylated hexaacyl disaccharide were firstdetermined; then these preferred buffers were evaluated with aphosphorylated hexaacyl disaccharide formulation including aphophatidylcholine.

TABLE 3 Stability of PHAD at 25° C. for 7 days in Select BuffersCondition/ Time Point Result 1 0.5 mg/ml PHAD in WATER 250° C./7 FAILdays 2 0.5 mg/ml PHAD in 10 mM 25° C./7 PASS trisodium citrate, pH 6.0days 3 0.5 mg/ml PHAD in 10 mM 25° C./7 PASS trisodium citrate, pH 5.0days 4 0.5 mg/ml PHAD in 50 mM 25° C./7 PASS trisodium citrate, pH 6.0days 5 0.5 mg/ml PHAD in 100 mM 25° C./7 FAIL trisodium citrate, pH 6.0days 6 0.5 mg/ml PHAD in 10 mM 25° C./7 FAIL sodium acetate, pH 6.0 days7 0.5 mg/ml PHAD in 10 mM 25° C./7 PASS disodium succinate, pH 6.0 days8 0.5 mg/ml PHAD in Phosphate 25° C./7 FAIL buffered saline (PBS) days 90.5 mg/ml PHAD in 200 mM Not precipitate Na₂HPO₄ and 100 mM applicablecitric acid, pH 6.0 10 0.5 mg/ml PHAD in 20 mM 25° C./7 PASS Na₂HPO₄ and10 mM citric acid, pH 6.0 days 11 0.5 mg/ml PHAD in 10 mM 25° C./7 PASSNa₂HPO₄, pH 6.0 days

The data in the table above demonstrate that citrate, succinate, andphosphate buffers from about 10 mM to 50 mM are superior to certainothers buffers examined, and provide stability at the listedtemperatures for the listed time periods.

TABLE 4 Stability of PHAD at 25° C. for 30 or 60 days and 37° C. for 7,60 days, or 4 months in Select Buffers Condition/ Time Point Result 0.5mg/ml PHAD in 20 mM 25° C./30 days FAIL Na₂HPO₄ and 10 mM citric acid,pH 6.0 0.5 mg/ml PHAD in Phosphate 25° C./30 days FAIL buffered saline(PBS) 0.5 mg/ml PHAD in 10 mM 25° C./30 days PASS trisodium citrate, pH5.0 0.5 mg/ml PHAD in 10 mM 25° C./30 days FAIL trisodium citrate, pH6.0 0.5 mg/ml PHAD in 10 mM 25° C./60 days FAIL Na₂HPO₄, pH 6.0 0.5mg/ml PHAD in 10 mM 25° C./60 days FAIL disodium succinate, pH 6.0 0.5mg/ml PHAD in 50 mM trisodium 25° C./60 days PASS citrate, pH 6.0 0.5mg/ml PHAD in 10 mM trisodium 37° C./7 days PASS citrate, pH 6.0 0.5mg/ml PHAD in 30 mM trisodium 37° C./7 days PASS citrate, pH 6.0 0.5mg/ml PHAD in 50 mM trisodium 37° C./7 days PASS citrate, pH 6.0 0.5mg/ml PHAD in 10 mM 37° C./7 days PASS Na₂HPO₄, pH 6.0 0.5 mg/ml PHAD in50 mM 37° C./7 days PASS Na₂HPO₄, pH 6.0 0.5 mg/ml PHAD in 10 mMtrisodium 37° C./7 days PASS citrate, 10 mM Na₂HPO₄, pH 6.0 0.5 mg/mlPHAD in 10 mM trisodium 37° C./60 days FAIL citrate, pH 6.0 0.5 mg/mlPHAD in 30 mM trisodium 37° C./60 days PASS citrate, pH 6.0 0.5 mg/mlPHAD in 50 mM trisodium 37° C./60 days PASS citrate, pH 6.0 0.5 mg/mlPHAD in 50 mM Na₂HPO₄, 37° C./60 days PASS pH 6.0 0.5 mg/ml PHAD in 10mM trisodium 37° C./60 days FAIL citrate, 10 mM Na₂HPO₄, pH 6.0 0.5mg/ml PHAD in 10 mM trisodium 37° C./4 FAIL citrate, pH 6.0 months 0.5mg/ml PHAD in 30 mM trisodium 37° C./4 PASS citrate, pH 6.0 months 0.5mg/ml PHAD in 50 mM trisodium 37° C./4 PASS citrate, pH 6.0 months 0.5mg/ml PHAD in 50 mM Na₂HPO₄, 37° C./4 PASS pH 6.0 months

The data in the table 4 demonstrate that citrate and phosphate buffersfrom about 30 mM to about 50 mM are superior to the other buffersexamined. The citrate and phosphate buffers provide stability at thelisted temperatures for the listed time periods. The data demonstratethe remarkable benefit of stability of increasing the citrate andphosphate concentrations to about 25 mM to about 50 mM, more preferably28 mM to about 50 mM, and most preferably 30 mM to about 50 mM. Thepreferred use of succinate as a buffer is shown from all of the datacollectively as described herein. Stability of phosphorylated hexaacyldisaccharide at 37° C. for 60 or more days in 30 mM to 50 mM citrate andphosphate buffers is unknown in the prior art and is an important aspectof the invention.

TABLE 5 Stability of a 0.5 mg/ml PHAD Formulation of the Invention inSelect Buffers and Select Phosphatidylcholines at 25° C. for 60 dayswithout Polysorbate 80 and without Extrusion. The phosphatidylcholineswere prepared at about a molar ratio of 2.5 to 1 to PHAD. Condition/Time Point Result PHAD formulation with DPPC in 25° C./60 days PASS 10mM trisodium citrate, pH 6.0 PHAD formulation with POPC in 25° C./60days PASS 10 mM trisodium citrate, pH 6.0 PHAD formulation with DMPC in25° C./60 days PASS 10 mM trisodium citrate, pH 6.0 PHAD formulationwith DPPC in 25° C./60 days PASS 50 mM trisodium citrate, pH 6.0 PHADformulation with DPPC in 25° C./60 days PASS 10 mM disodium succinate,pH 6.0 PHAD formulation with POPC in 25° C./60 days PASS 10 mM disodiumsuccinate, pH 6.0 PHAD formulation with DMPC in 25° C./60 days PASS 10mM disodium succinate, pH 6.0 PHAD formulation with DPPC in 25° C./60days PASS 10 mM Na₂HPO₄, pH 6.0

The data in the Table 5 demonstrate the remarkable and unexpectedbenefit of combining the citrate, succinate, and phosphate buffers atabout 10 mM to 50 mM with a phosphatidylcholine of the invention. Thecombination of the preferred buffers of the invention with aphosphatidylcholine results in superior stability of phosphorylatedhexaacyl disaccharide in the formulation compared to buffer alone.

TABLE 6 Condition/Time Point Result 1 Adjuvant formulation of Example 1except 25° C./14 days FAIL reconstituted in WATER, not extruded, 2-8°C./1 month PASS and without polysorbate 80 25° C./1 month FAIL 2-8° C./2months PASS 25° C./2 months FAIL 2 Adjuvant formulation of Example 1 (in25° C./7 days PASS trisodium citrate, pH 6.0 as stated 25° C./14 daysPASS above) not extruded and 25° C./1 month PASS without polysorbate 8025° C./2 months PASS 3 Adjuvant Formulation of Example 1 2-8° C./8months PASS (Lot 1214P69) 25° C./8 months PASS 4 Adjuvant Formulation ofExample 1 2-8° C./3 months PASS (Lot ENG-1) 25° C./2 months PASS 2-8°C./5 months PASS 25° C./4 months PASS 5 Adjuvant Formulation of Example1 2-8° C./3 months PASS 25° C./3 months PASS 2-8° C./6 months PASS 25°C./6 months PASS 6 FimCH Vaccine, 17 mM trisodium citrate, 2-8° C./4days PASS pH 5.4, consisting of: 25° C./4 days PASS 0.1 mg/ml adjuvantformulation of 2-8° C./3 months PASS Example 1 without polysorbate 802-8° C./4 months PASS 0.2 mg/ml FimCH, pH 5.4

As shown in Table 6, it is the combination of citrate at about 10 mMwith specific molar ratios of DPPC to phosphorylated hexaacyldisaccharide provides long-term stability at about 25° C.

As shown in the tables above, formulations prepared in water are stableshort-term when stored at 2° C. to 8° C.; however, the long sought-aftergoal is to reduce cold chain storage and management. The inventiveadjuvant formulation disclosed herein achieved this goal by providing anadjuvant formulation with extended stability at room temperature toabout 37° C. The data in Table 6 clearly show that the phosphorylatedhexaacyl disaccharide concentration of these formulations prepared incitrate buffer will remain stable for at least approximately 6 or moremonths at about 25° C. and potentially 2 to 3 years at 2° C. to 8° C.

The addition of citrate, succinate, or phosphate buffer to thePhosphatidylcholine:phosphorylated hexaacyl disaccharide formulationsremarkably and unexpectedly enables storage at room temperature andexposure up to and at about 37° C. Phosphatidylcholine:phosphorylatedhexaacyl disaccharide formulations prepared in water can produceequivalent immunogenic responses in mice and rabbits but are not stableat about 25° C.

Example 12

Particle sizes and zeta potentials were determined on PHAD formulationsusing Dynamic Light Scattering with the Malvern Zetasizer® ZS90 orBrookhaven Instruments Corp. using ZetaPlus Particle Sizing software.The manufacturer's directions and recommendations were followed. Table 7provides representative data. Zeta potential values are used as onepiece of qualitative data estimating the electrical charge at a bilayerand as described herein. Stability of the phosphorylated hexaacyldisaccharide formulations is experimentally determined from particlesize of the formulation and concentration of PHAD.

TABLE 7 Mean Effective Zeta Diameter Potential Sample (nm) (mV) 1Adjuvant formulation of Example 1, not 207 −56 extruded and withoutpolysorbate 80 2 Adjuvant formulation of Example 1, containing 309 −37DPPC:PHAD at a molar ratio of about 13:1, not extruded and withoutpolysorbate 80 3 Adjuvant formulation of Example 1, containing 293 −72DMPC and not DPPC, not extruded and without polysorbate 80 4 Adjuvantformulation of Example 1, containing 202 −76 DMPC:DLPC and not DPPC, inan approximate molar ratio with PHAD at 1.2:1.2:1 (DMPC:DLPC:PHAD) notextruded and without polysorbate 80 5 Adjuvant formulation of Example 1without 79 −46 polysorbate 80 6 Adjuvant formulation of Example 1 71 −407 Adjuvant formulation of Example 1, extruded at 185 −73 roomtemperature and without polysorbate 80 8 DPPC alone (no PHAD) asprepared in Example 1393 −6 1 without extrusion and without polysorbate80

As shown above, DPPC alone has a significantly less zeta potential andmuch larger mean particle size compared to the formulations of theinvention containing PHAD. The critical micelle concentration of DPPC isabout 0.46 nanomolar. These data offer evidence that DPPC alone is asignificantly different composition than a DPPC and phosphorylatedhexaacyl disaccharide composition.

Example 13

The particle sizes of phosphorylated hexaacyl disaccharide formulationsof Example 1 prepared with or without polysorbate 80 or glycerol werecompared when stored at 2° C. to 8° C. or about 25° C. to project(estimate) the stability of the particle sizes of the PHAD formulationsover 12 to 36 months. Multiple batches of phosphorylated hexaacyldisaccharide formulations were prepared and typically exhibitedparticles sizes between 70 to 100 nm immediately after extrusion. Thegoal for these phosphorylated hexaacyl disaccharide formulations is toensure that their mean effective diameter remains preferably less than150 nm over its shelflife, even more preferably less than 130 nm, whichis predicted to be 2 to 3 years or longer based upon the data describedherein. It is important to trend the mean effective diameter over acertain time period at intermediate/accelerated conditions, e.g. 25° C.,to assist with predicting the particle size at 2° C. to 8° C. inapproximately 2 or more years.

Particle sizes and zeta potentials were determined using Dynamic LightScattering with the Malvern Zetasizer® ZS90 or Brookhaven InstrumentsCorp. using ZetaPlus Particle Sizing software. The instrumentmanufacturer's directions and recommendations were followed.

TABLE 8 Mean Effective Time Point/ Diameter Sample Condition (nm)Adjuvant formulation of Example 1 without 1 month/25° C. 99 polysorbate80 Adjuvant formulation of Example 1 with 1 month/25° C. 104 0.1%polysorbate 80 Adjuvant formulation of Example 1 1 month/2-8° C. 79Adjuvant formulation of Example 1 1 month/25° C. 82 Adjuvant formulationof Example 1 with 1 month/25° C. 105 0.01% polysorbate 80 Adjuvantformulation of Example 1 without 4 months/2-8° C. 83 polysorbate 80Adjuvant formulation of Example 1 without 4 months/25° C. 114polysorbate 80 Adjuvant formulation of Example 1 with 4 months/2-8° C.94 0.1% polysorbate 80 Adjuvant formulation of Example 1 with 4months/25° C. 106 0.1% polysorbate 80 Adjuvant formulation of Example 1with 4 months/2-8° C. 90 0.01% polysorbate 80 Adjuvant formulation ofExample 1 with 4 months/25° C. 114 0.01% polysorbate 80 Adjuvantformulation of Example 1 4 months/25° C. 86 Adjuvant formulation ofExample 1 5 months/2-8° C. 82 Adjuvant formulation of Example 1 6months/25° C. 101 Adjuvant formulation of Example 1 6 months/2-8° C. 94Adjuvant formulation of Example 1 without 8 months/2-8° C. 117polysorbate 80

The data presented in Table 8 in this example project that the particlesizes of the phosphorylated hexaacyl disaccharide formulations stored at2° C. to 8° C. without polysorbate 80 will remain below 150 nm forapproximately a minimum of 2 years, and suggest potentially 3 years asdescribed below. In this specific example and only for the purpose ofthis test, stable means that the mean effective diameter remains below150 nm within the limitations of the instrument. To those skilled in theart, data from 25° C. is an intermediate/accelerated condition used toproject a shelf life at the long term storage condition of 2° C. to 8°C. These data clearly project that the particle sizes of these PHADformulations will remain stable for at least 6 or more months at about25° C. and potentially 3 years at 2° C. to 8° C. As described herein,this stability of these PHAD formulations at 25° C. is unknown in theprior art and unexpected.

Example 14

A Comparison of the Formulations of the Invention to Other AdjuvantFormulations at Inducing anti-FimH Antibodies in Mice.

The phosphorylated hexaacyl disaccharide formulation of the invention oraqueous formulation (Aqueous formulation in this example refers to themolar ratio of lipid to adjuvant described in U.S. Pat. No. 6,491,919and US Patent Application 20080131466) used below in this example wereprepared as described in Example 1 with the following exceptions. Thespecified lipid(s) and/or molar ratios (shown in parentheses) were usedand sonication occurred at about 45° C. for about 30 minutes to 2 hoursas needed to achieve a homogenous suspension. All lipids were purchasedfrom Avanti Polar Lipids as previously described herein.

Female C3H/HeN mice (approximately 9 weeks old) were purchased fromCharles River laboratories (Wilmington, Mass.). Mice were injected viathe intramuscular route (IM) in the right thigh under light isofluraneanesthesia (Henry Schein, Melville, N.Y.) in a 50 μl volume using a 30gauge needle. Mice were vaccinated with 12.5 μg phosphorylated hexaacyldisaccharide or Freund's adjuvant C (subcutaneous administration), and15 μg FimCH, on days 1 and 29. As known to those skilled in the art,when used, complete Freund's adjuvant was given on day 1 and incompleteFreund's adjuvant on day 29.

ELISA for serum antibody detection: Sera was collected from the mice atsacrifice and analyzed by ELISA for anti-FimH antibodies. FimH truncateT3 was adhered to Immulon 4 HBX plates (ThermoFisher) at 2 μg/ml in PBSovernight at 4° C. After washing with PBS+0.05% TWEEN 20 (polyethyleneglycol sorbitan monolaurate), open binding sites were blocked with 1.5%BSA (Sigma Aldrich) in PBS for 1 hr. After washing, dilutions of samplesera (in PBS with 0.05% TWEEN 20 (polyethylene glycol sorbitanmonolaurate), 0.1% BSA, 0.5% methyl α-D-mannopyranoside) were incubatedfor 2 hrs. After washing, 1:500 diluted biotinylated Goat anti-Mouse IgGdetection antisera (Sigma Aldrich) in sample dilution buffer wasincubated in the wells overnight at 4° C. After washing, 1:25,000diluted Avidin-Horse Radish Peroxidase (HRP, Sigma Aldrich) in sampledilution buffer was incubated in the wells for 20 minutes. Afterwashing, HRP activity was detected using TMB substrate in Phosphocitratebuffer (Sigma Aldrich). Optical density was read at 630 nm using aVERSAMAX PLUS microplate reader and analyzed using SOFTMAX Pro analysissoftware (Molecular Devices, Sunnyvale, Calif.). Antibody titer wasdefined as the highest dilution with signal above background.

TABLE 9 Antibody Dilution Adjuvant / Formulation Titers No Adjuvant; N =6 mice 1:20,000 Experiment 1: Reference Adjuvant: 1:200,000 Freund'sadjuvant; N = 6 mice Experiment 2: Reference Adjuvant: 1:200,000Freund's adjuvant; N = 7 mice Experiment 1: DPPC:PHAD (1:3.9) 1:200,000aqueous formulation; N = 8 mice Experiment 2: DPPC:PHAD (1:3.9)1:200,000 aqueous formulation; N = 10 mice DPPC:PHAD (2.6:1) adjuvantformulation; 1:400,000 N = 10 mice DPPC:PHAD (13:1) adjuvantformulation; 1:400,000 N = 10 mice DPPC:DPPG:PHAD (2.6:0.3:1) adjuvant1:400,000 formulation; N = 10 mice DPPC:DPPG:PHAD (2.6:2.6:1) adjuvant1:400,000 formulation; N = 10 mice

As clearly shown in Table 9, a molar ratio of DPPC to phosphorylatedhexaacyl disaccharide of about 2:1 to about 13:1 was superior atenhancing an immune response to FimH in mice when compared to noadjuvant, Freund's adjuvant, or the aqueous formulation of DPPC tophosphorylated hexaacyl disaccharide at a molar ratio of 1:3.9. Freund'sadjuvant is considered a standard adjuvant used in animal experiments.The data shows that the molar ratio of the invention described herein ofabout 2:1 to 13:1 is superior to this frequently used preclinicaladjuvant. This is one aspect of the invention that demonstrates itssuperiority to the prior art formulations. The data in Table 9 alsodemonstrate that adding DPPG into these formulations does not diminishthe intended use of the phosphorylated hexaacyl disaccharideformulations, i.e., to enhance an immune response to FimH.

Example 15

Determination that the Formulations of the Invention remain a HomogenousMixture within 24 Hours. The procedure of Example 1 was used to preparethe phosphorylated hexaacyl disaccharide formulation with a molar ratioof DPPC:phosphorylated hexaacyl disaccharide at about 2.5 to 1, exceptpolysorbate 80 was not added. The vial containing the phosphorylatedhexaacyl disaccharide formulation was gently inverted about three tofive times. Then, the phosphorylated hexaacyl disaccharide formulationwas allowed to remain at room temperature for about 24 hours. After 24hours and without inverting, shaking, or stirring the phosphorylatedhexaacyl disaccharide formulation, small aliquots were carefully removedfrom the top, middle, and bottom of the phosphorylated hexaacyldisaccharide suspension. These aliquots were analyzed for phosphorylatedhexaacyl disaccharide concentration via HPLC as previously describedherein. The results demonstrated that the aliquots from the top, middle,and bottom contained equivalent phosphorylated hexaacyl disaccharideconcentrations. These results demonstrate the phosphorylated hexaacyldisaccharide formulation of the invention does not settle within 24hours.

Example 16

Formulations of the Invention used in Human Clinical Study.

The adjuvant formulation of Example 1 and FimCH were prepared under cGMPfor use in a human clinical study involving females of about 21 to 64years of age. A vaccine of FimCH and phosphorylated hexaacyldisaccharide formulation was prepared by adding a predetermined volumeof a phosphorylated hexaacyl disaccharide formulation to a vial of FimCHas previously described herein to obtain the preferred concentrations ofFimCH and phosphorylated hexaacyl disaccharide per each vial. Anappropriate volume of the prepared vaccine was injected IM(intramuscular) to administer either 50 μg or about 107 μg of FimCH witheither 10 μg, 20 μg, or about 40 μg of phosphorylated hexaacyldisaccharide to each female subject.

From these IM injections in humans, the vaccine demonstrates that theadjuvant formulation of the invention with FimCH produces less severeinjection site and systemic reactions in humans compared to certainother known adjuvant formulations used in humans. This is a remarkableaspect of the invention. Interim Data from Injection Site and SystemicReactions are shown below for number of injections as of the interimanalysis per female. The human study is ongoing and the females in thestudy are scheduled to receive 4 injections.

TABLE 10 FimCH / Severe Injection Number of PHAD dose Number of Site andFemales in micrograms Injections Systemic Reactions Group 1 5 107 pg/0μg 3 to 4 None Group 2 8 50 pg/10 μg 2 to 3 None Group 3 16 50 pg/20 μg1 to 2 None Group 4 8 50 pg/40 μg 1 None

At the time of this interim analysis, more than 60 IM injections to 37females had been made and no severe injection site and systemicreactions have been observed. At the time of this interim analysis,females in Group 2 have demonstrated an antibody response to FimH aftertwo IM injections greater than 10-fold from initial values prior tovaccination. These data demonstrate the vaccine is producing theintended antibody response against FimH. Females in this study willreceive up to 4 IM injections of the vaccine. Groups 5 and 6 will openenrollment for females with a history of recurrent UTI.

In comparison, administration of the Cervarix vaccine that contains theadjuvants MPL with alum results in approximately 8% to more than 10% ofsubjects exhibiting severe injection site reactions and systemicreactions. As reported in the publication of Treanor et al. (Vaccine,2013, 31(48), 5760-5), phosphorylated hexaacyl disaccharide has beenprepared in different formulations with squalene and administered tohumans. Per this report, 5 μg of phosphorylated hexaacyl disaccharide inthis formulation resulted in severe local reactions, chills, and rigorand was so limiting that use of this formulation in future studies onlycontained 2 or less micrograms of PHAD. At 1 micrograms ofphosphorylated hexaacyl disaccharide in this formulation, severeinjection site reactions and systemic reactions were still observed. Inthis formulation of Treanor et al. at 2 or less micrograms ofphosphorylated hexaacyl disaccharide, the benefits of the adjuvantphosphorylated hexaacyl disaccharide producing an immune response arelimited thereby robbing the potential of phosphorylated hexaacyldisaccharide due to an inferior formulation. However, the adjuvantformulation of the invention overcomes this limitation with superiorformulations that enable administration of up to 50 μg of phosphorylatedhexaacyl disaccharide, or potentially more, with less severe injectionsite and systemic reactions. These human data offer evidence thatphosphorylated hexaacyl disaccharide can be administered to humans at100 μg or more micrograms with the formulations described herein. Theadjuvant formulations of the invention are superior to thoseformulations previously attempted.

All references, including without limitation all papers, publications,presentations, texts, reports, manuscripts, brochures, internetpostings, journal articles, periodicals, and the like, cited in thisspecification are hereby incorporated by reference. The discussion ofthe references herein is intended merely to summarize the assertionsmade by their authors and no admission is made that any referenceconstitutes prior art. The inventors reserve the right to challenge theaccuracy and pertinence of the cited references.

It is intended that all patentable subject matter disclosed herein beclaimed and that no such patentable subject matter be dedicated to thepublic. Thus, it is intended that the claims be read broadly in light ofthat intent. In addition, unless it is otherwise clear to the contraryfrom the context, it is intended that all references to “a” and “an” andsubsequent corresponding references to “the” referring back to theantecedent basis denoted by “a” or “an” are to be read broadly in thesense of “at least one.” Similarly, unless it is otherwise clear to thecontrary from the context, the word “or,” when used with respect toalternative named elements is intended to be read broadly to mean, inthe alternative, any one of the named elements, any subset of the namedelements or all of the named elements.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results obtained. Itshould be understood that the aforementioned embodiments are forexemplary purposes only and are merely illustrative of the many possiblespecific embodiments that can represent applications of the principlesof the invention. Thus, as various changes could be made in the abovemethods and compositions without departing from the scope of theinvention, it is intended that all matter contained in the abovedescription as shown in the accompanying drawings shall be interpretedas illustrative and not in a limiting sense.

Moreover, one of ordinary skill in the art can make various changes andmodifications to the invention to adapt it to various usages andconditions, including those not specifically laid out herein, withoutdeparting from the spirit and scope of this invention. Accordingly,those changes and modifications are properly, equitably, and intended tobe, within the full range of equivalents of the invention disclosed anddescribed herein.

The invention claimed is:
 1. A composition comprising an adjuvantformulation comprising phosphorylated hexaacyl disaccharide and a bufferselected from the group consisting of citrate and succinate from 15 mMto about 50 mM.
 2. The composition of claim 1, wherein the bufferconcentration is from 20 mM to about 50 mM.
 3. The composition of claim1, wherein the buffer concentration is from about 30 mM to about 50 mM.4. The composition of claim 1, wherein the composition comprisesphosphatidylcholine.
 5. The composition of claim 1, wherein thecomposition comprises phosphatidylcholine, and whereinphosphatidylcholine is selected from the group consisting of1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).
 6. The composition ofclaim 1, wherein the composition comprises phosphatidylcholine, andwherein phosphatidylcholine is selected from the group consisting ofDMPC, DPPC, and DSPC.
 7. The composition of claim 4, wherein thecomposition comprises phosphatidylcholine at a molar ratio withphosphorylated hexaacyl disaccharide at about 1:1 to about 20:1.
 8. Thecomposition of claim 5, wherein the composition comprisesphosphatidylcholine at a molar ratio with phosphorylated hexaacyldisaccharide at about 1:1 to about 20:1.
 9. The composition of claim 6,wherein the composition comprises phosphatidylcholine at a molar ratiowith phosphorylated hexaacyl disaccharide at about 1:1 to about 20:1.10. The composition of claim 4, wherein the composition comprisesphosphatidylcholine at a molar ratio with phosphorylated hexaacyldisaccharide at about 2:1 to about 5:1.
 11. The composition of claim 5,wherein the composition comprises phosphatidylcholine at a molar ratiowith phosphorylated hexaacyl disaccharide at about 2:1 to about 5:1. 12.The composition of claim 5, wherein phosphorylated hexaacyl disaccharideis comprised of


13. The composition of claim 1, wherein the composition comprises aneffective amount of FimCH.
 14. The composition of claim 1, wherein thebuffer is citrate.
 15. The composition of claim 1, wherein the buffer issuccinate.
 16. The composition of claim 1, wherein the composition isstable when exposed to room temperature for 60 or more days.
 17. Thecomposition of claim 1, wherein the composition is stable when exposedto room temperature for 6 or more months.
 18. The composition of claim1, wherein the composition is stable when exposed to about 37° C. for 2or more months.
 19. The composition of claim 1, wherein the compositionis an aqueous buffered suspension with a mean particle size of 150nanometers or less.
 20. The composition of claim 1, wherein thecomposition is essentially free of a second adjuvant.
 21. Thecomposition of claim 1, wherein the composition is substantially free ofsaline.
 22. The composition of claim 1, wherein the composition containsless than 10 mM of NaCl.
 23. The composition of claim 1, wherein thecomposition is essentially free of NaCl.
 24. The composition of claim 1,wherein the composition is substantially free of cholesterol.
 25. Thecomposition of claim 1, wherein phosphorylated hexaacyl disaccharide iscomprised of


26. The composition of claim 1, wherein the buffer concentration is from20 mM to about 50 mM, and the composition further comprisesphosphatidylcholine at a molar ratio with phosphorylated hexaacyldisaccharide at about 2:1 to about 5:1, and phosphatidylcholine isselected from the group consisting of DMPC, DPPC, and DSPC, wherein thecomposition is an aqueous buffered suspension with a mean particle sizeof 150 nanometers or less and essentially free of cholesterol.
 27. Amethod for administering a vaccine composition to a human comprising thesteps of: administering to the human a vaccine composition comprising anadjuvant formulation comprising phosphorylated hexaacyl disaccharide anda buffer selected from the group consisting of citrate and succinatefrom 15 mM to about 50 mM.
 28. The method of claim 27, wherein thevaccine composition is essentially free of a second adjuvant.
 29. Themethod of claim 27, wherein phosphorylated hexaacyl disaccharide iscomprised of